5-HT1A receptor agonists are thought to enhance the antipsychotic-like effects of dopamine D2 receptor antagonists while reducing their potential to produce extrapyramidal side effects. Thus, 5-HT1A receptor agonist properties of mixed 5-HT1A receptor agonists/D2 receptor antagonists might be of clinical importance. The antipsychotics, clozapine and nemonapride, and the putative antipsychotic, bromerguride, have intermediate to high affinity for 5-HT1A receptors. The present study examined the 5-HT1A receptor agonist activity of nemonapride and bromerguride, in comparison with clozapine, which has partial 5-HT1A receptor agonist properties in vitro. Here, 5-HT1A receptor activation was examined in vitro, by measuring forskolin-stimulated cAMP accumulation in HeLa cells expressing human 5-HT1A receptors, and in vivo, by using microdialysis to measure the extracellular concentration of hippocampal 5-hydroxytryptamine (5-HT) in rats. Nemonapride markedly decreased both forskolin-stimulated cAMP accumulation and the extracellular concentration of 5-HT; both effects were antagonized by the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY100635). In contrast, clozapine only partially decreased forskolin-stimulated cAMP accumulation and extracellular 5-HT, and only its effects on cAMP accumulation were attenuated by WAY100635. Bromerguride decreased neither forskolin-stimulated cAMP accumulation nor extracellular 5-HT; instead, it antagonized the decrease of cAMP accumulation produced by 5-HT and the decrease of extracellular 5-HT produced by the 5-HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The selective D2 receptor antagonist, raclopride, affected neither forskolin-stimulated cAMP in vitro nor extracellular 5-HT in vivo. Thus, in contrast with clozapine and bromerguride, only the novel antipsychotic, nemonapride, exhibited marked 5-HT1A receptor agonist properties both in vitro and in vivo; conceivably, these properties may play a role in its preclinical and clinical effects.