The activity of serotonin (5-HT) receptor agonists, partial agonists and antagonists, and various other neurotransmitter receptor antagonists at human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells was investigated. 5-HT1A receptor-mediated inhibition of adenylate cyclase was studied by measuring inhibition of cAMP accumulation, induced by forskolin. At 100 microM forskolin produced a 100-fold increase in cAMP formation: 5-HT concentration dependently inhibited the cAMP formation; maximal inhibition was attained at 1 microM 5-HT and represented 90% of the stimulated cAMP formation. Full inhibition was observed with 5-HT1A receptor agonists: N,N-dipropyl-8-hydroxy-2-aminotetralin (8-OH-DPAT) and flesinoxan, and non-selective 5-HT receptor agonists: d-lysergic acid diethylamide (d-LSD), RU 24,969, bufotenine, methysergide and tryptamine. The rank order of potency of the compounds for inhibiting the cAMP formation corresponded to the rank order of the binding affinities of the drugs for the 5-HT1A receptor. Partial inhibition was obtained with submicromolar concentrations of buspirone, spiroxatrine and ipsapirone. A slight inhibition was observed with 1 microM 5-HT receptor agonist CP 93129 and 1 microM 5-HT receptor antagonists mesulergine and BW-501. No inhibition was found with: the 5-HT receptor agonists quipazine, sumatriptan and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); the 5-HT receptor antagonist ICS-205,930; and other neurotransmitter receptor antagonists such as pindolol, CGP 20712-A, prazosin, sulpiride and pyrilamine. Spiperone and pindolol fully antagonized the agonist-mediated inhibition of forskolin-stimulated cAMP formation. Partial inhibition of the agonist-mediated inhibition of forskolin-stimulated cAMP formation was apparent with 1 microM ocaperidone and 1 microM ipsapirone. It can be concluded that HeLa cells, permanently expressing human 5-HT1A receptors, are a valid cellular system for studying the negative coupling of 5-HT1A receptors to adenylate cyclase and the action of compounds thereupon.