The 5-Hydroxytriptamine 1A receptor (5-HT1A) is expressed both as a pre- and post-synaptic receptor in neurons. The presynaptic receptor preferentially desensitizes compared to post-synaptic receptors, suggesting different underlying mechanisms of agonist-induced desensitization. Using F11 cells as a model of post-synaptic neurons, the present study examined the role of protein kinase C (PKC) and protein kinase A (PKA) in desensitization of the 5-HT1A-receptor by agonist. Desensitization in whole cell experiments was dependent on internal [Ca2+] and was blocked by chelation of intracellular Ca2+. Using the perforated patch technique, desensitization was reduced when Ba2+ was used as the conducting cation. Selective inhibitors of conventional PKC isoforms prevented 5-HT-induced desensitization, whereas an inhibitor of PKA did not. In cells in which 3 PKC/PKA sites located in the third intracellular loop (i3) of the 5-HT1A receptor were mutated (i3, T229A-S253G-T343A), 5-HT-mediated desensitization was reduced (and abolished in the absence of intracellular Ca2+). In cells in which a fourth mutation was added (T149 in the second i2 loop), the cells responded similarly to the triple mutants suggesting that phosphorylation of T149 does not contribute greatly to the desensitization induced by 5-HT-mediated activation of PKC. Thus agonist-induced uncoupling of the 5-HT1A-receptor is PKC-dependent, but requires a different set of phosphorylation sites than phorbol ester-mediated PKC activation, suggesting differential recruitment of PKC. Furthermore, these studies reveal that 5-HT1A-receptor desensitization utilizes a different kinase in F11 cells and serotonergic neurons, which may in part account for their differential sensitivity in vivo.