Abstract: Currently, a search for selective antagonists of luteinizing hormone (LH) receptor, which are required to suppress the steroidogenesis in hormone-dependent tumors and to prevent the ovarian hyperstimulation syndrome, is carried out. One approach to tackle this problem is the development of low-molecular-weight antagonists of the allosteric site of this receptor, which is located in its transmembrane domain. The aim of this work was to develop the heterocyclic compounds, the derivatives of thieno[2,3-d]pyrimidine (TP31), pyrimido[4,5,6-de][1,6]naphthyridine (PP10), and pyrido[3,4-d]pyrimidine (PP17), and to study their ability to affect the functional activity of the LH receptor in the in vitro and in vivo conditions. It was shown that TP31 at micromolar concentrations suppressed stimulating effects of human chorionic gonadotropin (hCG) and TP03, an allosteric agonist of the LH receptor, on the adenylyl cyclase activity in rat testicular membranes; its effect was most pronounced in relation to the stimulating effects of TP03. This was due to a higher selectivity of the TP31 antagonist with respect to the cAMP-dependent signaling cascades, predominantly activated by TP03 and realized through G_s proteins. PP17 inhibited stimulatory effects of hCG and TP03 on the adenylyl cyclase activity to a similar extent but was less active compared to TP31. Upon intratesticular (10 mg/kg) or intraperitoneal (45 mg/kg) administration to male rats, TP31 and PP17 decreased the baseline plasma level of testosterone and inhibited the testosterone production stimulated by hCG (100 IU/rat); the inhibitory effect of TP31 was much more pronounced than that of PP17. PP10 exhibited a weaker antagonistic activity than TP31 and PP17 in the in vitro and in vivo conditions. The data obtained indicated that TP31, the most active functional antagonist among the studied compounds, binded to the allosteric site of the LH receptor, made it less accessible to allosteric agonists and impairs the hormonal signal transduction through the LH receptor. This suggests the prospects of the development of TP31-based inhibitors of LH-dependent pathways and steroidogenesis.