In order to clarify the mechanism of action of the putative nonbenzodiazepine anxiolytic SM-3997 [3a alpha,4 beta,7 beta,7a alpha)-Hexahydro-2-(4-(4-(2-pyrimidinyl)-1- piperazinyl)-butyl)-4,7-methano-1H-isoindole-1,3 (2H)-dione dihydrogen citrate), in vitro binding studies with radiolabeled compound were performed. 3H-SM-3997 bound rapidly, reversibly and in a saturable manner with high affinity to rat brain hippocampal membranes (Kd = 9.4 nM, Bmax = 213 fmol/mg protein). This specific binding was displaced by 5-hydroxytryptamine (5-HT) and related compounds. Especially, 8-OH-DPAT, a 5-HT-1A selective agonist, bound with the highest affinity to these binding sites. 3H-SM-3997 binding, however, was not displaced by a variety of other neurotransmitters, neuropeptides and some other drugs. EDTA and physiological concentration of Na+ inhibited this specific binding, but several divalent cations, Mn2+, Ca2+ and Mg2+, enhanced this binding. GTP decreased the affinity of these binding sites for 3H-SM-3997 without changing the number of binding sites, but GMP and ATP did not influence 3H-SM-3997 binding. Furthermore, 3H-SM-3997 bound with marked regional selectivity to hippocampal membranes. These characteristics and the regional distribution of 3H-SM-3997 binding sites were very similar to those of 3H-8-OH-DPAT binding sites (5-HT-1A receptors). Therefore, these results indicate that SM-3997 binds selectively and with high affinity to 5-HT-1A receptors in rat brain and may be an agonist.