INTRODUCTION: Atropine and scopolamine are two well-studied 'classical' muscarinic cholinergic antagonists, with multiple actions in the peripheral and central nervous systems, and various side-effects. They potently affect human and animal behavior, inducing specific 'deliriant' hallucinations, affecting anxiety levels, memory and general locomotion. However, their exact psychopharmacological profiles remain complex and poorly understood, necessitating further preclinical testing of these CNS drugs and related compounds. Zebrafish (Danio rerio) are rapidly emerging as a powerful tool for psychopharmacology research. Here, we utilize zebrafih behavioral testing procedures to characterize acute neuroactive effects of atropine and scopolamine. METHODS: Behavioral testing was performed in adult wild-type outbred short-fin zebrafish between 11.00 and 17.00 h, using tanks with water adjusted to the holding room temperature, to assess zebrafish behavior in the novel tank test. Prior to testing, fish were preexposed in a 0.5-L plastic beaker for 20 min to either drug-treated or drug-free water. For treatments, fish were randomly divided in 4 groups (n = 15): drug-free control, 30 mg/L, 90 mg/L and 120 mg/L for atropine (Experiment 1); and 60 mg/L, 120 mg/L and 180 mg/L and 240 mg/L for scopolamine (Experiment 2). RESULTS AND DISCUSSION: Acute atropine strongly affected adult zebrafish locomotor behavior, as fish treated with 90 mg/L covered significantly longer distance than did control group and other groups tested (2374±71 cm vs. 1761±95 cm, p<0.005 by Dunn's post-hoc test for significant Kruskal-Wallis data). Compared to control fish, the maximal swimming velocity was also significantly higher in the 90 mg/L group (7.9 ±0.24 cm/s vs. 5.9±0.32 cm/s control, p<0.003). Finally, fish from the 120 mg/L cohort began to show increased minimum acceleration (1051±400 cm/s2 vs. 714±351 cm/s2 control, p<0.05), which was not observed in normal control zebrafish. In contrast, there was no significant difference between control and treated with atropine groups in meander, latency to the top, mean plot of not moving, top time or top zone transition endpoints. Thus, treatment with atropine lead to increased swimming velocity and distance covered, suggesting that atropine at doses tested may possesses hyperlocomotor 'deliriant' CNS properties. Acute scopolamine affected adult zebrafish behavior as well, as fish treated with 120 and 240, but not with 180 mg/L, have significantly decreased top zone transitions than did control group (12.5±1.9 n vs. 21.0±1.6 n control, p<0.01 and 12.6±1.7 n vs. 21.0±1.6 n control, p<0.01 respectively). The latency to enter the top was also significantly longer in the 120 mg/L, but not in the 180 mg/L and 240 mg/L group (94.4±14.6 s vs. 40.3±6.2 s control, p<0.05). Decreased number of entering the top was seen in fish treated with both 180 and 240 mg/L (26.1±1.7 n vs. 33.4±2.3 n control, p<0.01 and 20.4±3.0 n vs. 33.4±2.3 n control, p<0.01 respectively), whereas no changes were observed in locomotor endpoints (distance traveled, velocity). Fish from the 120 mg/L cohort began to show increased minimum acceleration (-50.4±24.0 cm/s2 vs. -748.4±58.1 cm/s2 control, p<0.001). Collectively, this profile suggests anxiogenic and/or mild psychostimulant simulative properties of scopolamine in zebrafish, which may be relevant to the drug's known clinical delirium-inducing effects. While further research is needed to dissect in detail the complex behavioral effects of these two compounds in zebrafish, the overall phenotypic vector of overlapping changes observed here, and the sensitivity of fish to both drugs tested, support their growing utility as powerful biological sensors and screens for hallucinogenic/psychotropic drugs.