The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT_1A receptors to N-type Ca²⁺ channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 ± 6.8 % 5-HT-mediated, but not GTP--S-induced, inhibition of Ca²⁺ current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP--S. 5-HT_1A receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT_1A receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca²⁺ channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT_1A receptors, with only a 13.4 ± 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT_1A receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-8-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT_1A receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca²⁺ channel.