The relaxin family peptide receptors RXFP1 and RXFP2 are highly similar receptors that share approximately 80% amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, which is important for relaxin-mediated decidualization and myometrial inhibition. Despite the high homology, the receptors couple to different G-proteins to affect cAMP accumulation. This study aimed to determine the region of RXFP1 that directs coupling to the delayed Galpha(i3) pathway by using receptor mutagenesis. Receptor chimeras suggested that activation of this pathway by RXFP1 was dependent upon the membrane-anchored domain of the receptor. Further receptor mutagenesis showed that activation of the Galpha(i3)-Gbetagamma-PI3K-PKCzeta cAMP pathway by RXFP1 is dependent upon the C-terminal 10 amino acids of the receptor and absolutely requires Arg(752).