Relaxin is a two-chain hormone, structurally similar to insulin, that mediates pleiotropic effects in various physiological systems. The recent discovery of the human gene 2 (H2) relaxin receptor, RXFP1 (Hsu et al., 2002), and the related insulin-like peptide 3 (INSL3) receptor, RXFP2 (Kumagai et al., 2002), identified two G-protein-coupled receptors that cause cAMP accumulation. We recently identified differential G-protein coupling of these receptors to cAMP: both receptors couple to Gαs and GαoB, which increase and decrease cAMP accumulation respectively (Halls et al., 2006); but only RXFP1 recruits Gαi3 with time to further increase cAMP via a Gβγ-PI3K-PKCζ pathway (Halls et al., 2006; Nguyen et al., 2005). This study examined the mechanism of differential G-protein coupling using an α-screen cAMP accumulation assay. C-terminal tail truncates were generated for both RXFP1 (tRXFP1-703) and RXFP2 (tRXFP2-712). cAMP accumulation characteristics of tRXFP2-712 did not differ from RXFP2 (30min INSL3 (30nM) response as % forskolin (100μM) (n): RXFP2 5.2±1.9(4), pertussis toxin (PTX) 11.2±2.3(4), P<0.01; tRXFP2-712 4.5±0.8(6), PTX 9.8±0.5(5), P<0.001). However, tRXFP1-703 displayed lost coupling to Gαi3 and the Gβγ-PI3K-PKCζ pathway, instead becoming ‘RXFP2-like’ (30min H2 relaxin (30nM) response as % forskolin (100μM) (n): RXFP1 20.5±2.8(5), PTX 10.9±1.7(6), P<0.001; tRXFP1-703 6.4±1.6(9), PTX 10.8±0.4(8), P<0.001). Similarly, truncation of the final 10 C-terminal amino acids of RXFP1 (tRXFP1-747) also switched behaviour (tRXFP1-747 9.9±3.3(5), PTX 20.6±4.8(5), P<0.01). Finally, the production of RXFP1/RXFP2 chimeras with exchanged receptor transmembrane regions caused a loss of the delayed pathway signalling for RXFP1 (RXFP1/2 6.8±1.8(5), PTX 13.2±2.8(6), P<0.01), but a gain of this pathway for RXFP2 (RXFP2/1 17.6±5.3(7), PTX 9.0±1.6(7), P<0.01). Thus the final 10 residues of the C-terminal tail of RXFP1 enable coupling to the Gβγ-PI3K-PKCζ pathway to cause the delayed phase of cAMP accumulation.