The relaxin family peptide receptors (RXFP) 1 and 2 are targets for the relaxin family peptides relaxin and insulin-like peptide 3 (INSL3), respectively. Although both receptors and peptides share a high degree of sequence identity, the cAMP signaling pathways activated by the two systems are quite distinct. Relaxin activation of RXFP1 initially results in accumulation of cAMP via G(alpha)(s), but this is modulated by inhibition of cAMP through G(alpha)(oB). Over time, RXFP1 recruits coupling to G(alpha)(i3), causing additional cAMP accumulation via a G(alpha)(i3)-Gbetagamma-phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC)zeta pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via G(alpha)(s), modulated by cAMP inhibition through G(alpha)(oB). Thus, the aim of this study was to identify the cause of differential G-protein coupling between these highly similar receptors. Construction of chimeric receptors revealed that G(alpha)(i3) coupling is dependent upon the transmembrane region of RXFP1 and independent of the receptor ectodomain or ligand bound. Generation of C-terminal truncated receptors identified the terminal 10 amino acids of the RXFP1 C terminus as essential for G(alpha)(i3) signaling, and point mutations revealed an obligatory role for Arg(752). RXFP1-mediated G(alpha)(i3), but not G(alpha)(s) or G(alpha)(oB), signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to G(alpha)(i3) after only 3 min of receptor stimulation. Therefore, RXFP1 coupling to the G(alpha)(i3)-Gbetagamma-PI3K-PKCzeta pathway requires the terminal 10 amino acids of the RXFP1 C terminus and membrane raft localization, and the observed delay in this pathway occurs downstream of G(alpha)(i3).