P11 cells were transfected with DNA for the human 5-hydroxytryptamine1A (5-HT1A) receptor. These cells stably expressed the 5-HT1A receptor coupled to the inhibition of adenylyl cyclase, and not to the stimulation of phosphoinositide hydrolysis. Homologous and heterologous regulation of the 5-HT1A receptor was studied in this cell system. Pretreatment of P11-5HT1A cells with the 5-HT1 receptor agonist 5-carbox-amidotryptamine (5-CT) resulted in a 3-fold increase in both basal and forskolin-stimulated cAMP accumulation, and desensitization of the 5-HT1A receptor as indicated by a decrease in the potency of 8-hydroxydipropylaminotetralin (8-OH-DPAT) to inhibit forskolin-stimulated cAMP accumulation (vehicle-treated cells: EC50 = 2.3 +/- 0.8 nM; 5-CT-treated cells: 9.9 +/- 0.4 nM). The sensitization of adenylyl cyclase as a result of chronic agonist exposure was prevented by the 5-HT1A antagonist WAY100635, which indicated that the effect of 5-CT pretreatment on basal and forskolin-stimulated cAMP accumulation was mediated by 5-HT1A receptor activation. Pretreatment of cells with pertussis toxin abolished the inhibition of forskolin-stimulated cAMP accumulation mediated by 5-HT1A receptor activation and prevented the sensitization of adenylyl cyclase as a result of chronic 5-HT1A receptor agonist exposure. Pretreatment of P11-5HT1A cells with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), also resulted in desensitization of the 5-HT1A receptor, as indicated by a marked decrease in the potency and intrinsic activity of 8-OH-DPAT. No change in the binding characteristics (i.e., Kd or Bmax) of [3H]8-OH-DPAT to 5-HT1A receptor sites was observed after 5-CT or PMA treatments. Activation of alpha-1 adrenergic receptors, but not 5-HT2A receptors, had effects on 5-HT1A receptor responsiveness similar to those seen with PMA pretreatment. In P11-5HT1A cells, homologous regulation of the 5-HT1A receptor was characterized by sensitization of adenylyl cyclase and a decrease in agonist potency, whereas heterologous regulation of the 5-HT1A receptor was characterized by a greater decrease in agonist potency, as well as a marked decrease in intrinsic activity.