The 5-hydroxytryptamine1A receptor (5-HT1AR) is a G-protein-coupled receptor negatively coupled to adenylyl cyclase (AC). We have studied the functional domains of 5-HT1AR using synthetic peptides to block or mimic receptor function. The entire second intracellular loop (5-HT1AR-i2) and the carboxyl end of the third intracellular loop (5-HT1AR-i3-C) strongly inhibited forskolin-stimulated AC activity. These effects were not additive with those of 5-HT. Like 5-HT, the peptides 5-HT1AR-i3-C and -i2 weakly inhibited AIF4- and Mn2+ stimulated AC activity. 5-HT1AR binding assays indicated that peptides could interact with the same G-protein pool as the 5-HT1AR. 5-HT1AR-i3-C- and -i2-stimulated [35S]guanosine 5'-O-(thiotriphosphate) binding on Go/Gi proteins. Only 5-HT1AR-i3-C partially adopted an alpha-helical conformation in solution. These data show that different domains in the 5-HT1AR second and third intracellular loops can couple to and activate Gi proteins in order to mediate AC inhibition. Peptide-induced AC inhibition was not sensitive to pertussis toxin as opposed to the 5-HT1AR-mediated effect. Our data show that the 5-HT1AR and the 5-HT1AR peptides activate Gi proteins in a slightly different manner.