Results of behavioral tests made postflight after two independent travels of Drosophila melanogaster to International Space Station (ISS) are presented. In Experiment 1, the first instar larvae were launched, and the eclosed imagoes gave the next generation that developed from the egg at ISS. After return to Earth the behavior of adult males of the next generation was examined. In Experiment 2, the newly emerged adult males were launched and after 7.5 days of staying at ISS were tested in the lab. In Experiment 1, we found pronounced influence of development in Space on subsequent locomotor behavior of adult flies. In flies developed in Space, all three independent parameters of locomotor behavior (run frequency, run duration and running speed) were increased, at least at certain time points, during the first hour of registration. At the same time, the climbing ability of experimental flies was reduced. In Experiment 2, no changes in the climbing ability were detected. During the first 30 min after registration beginning the flown flies performed less frequent but more extended runs than the control flies did. However, later the flown flies demonstrated an increased run frequency. Additionally, the interpulse interval in the pulse courtship song was shorter in the flown males. Taken together, the results point to a considerable activation of the central nervous system in flies developed at the ISS and to a minor one in the adult flies travelled to the ISS. This shows that earlier stages of development are more sensitive to spaceflight factors than the adult stage.