In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine_1A receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged α_i3 subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1A-mediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G_i-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of Gα_i protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca²⁺ concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Gα_i3-CFP relocation and changes in [Ca²⁺]_i upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.