The receptors for H2 relaxin and insulin-like peptide 3, relaxin family peptide receptor (RXFP) 1 and RXFP2, respectively, were recently identified, but their signaling pathways are not yet well characterized. Although previous work has suggested that cAMP is a major signaling pathway activated by these receptors, RXFP1 has also been shown to activate a number of other signaling proteins. To this end, we examined the effect of stimulation of RXFP1 and RXFP2 receptors [expressed in human embryonic kidney (HEK) 293T cells] with human relaxin family peptides on a number of transcription factor-response elements coupled to reporter genes. Hence, reporter gene activity measured by enzyme activity in the cell media is a measure of the activation of a particular signaling pathway. Eight reporter genes were tested at both receptors as a screen to identify other signaling pathways activated by RXFP1 and RXFP2. The cAMP-response element reporter was strongly activated by both receptors. This effect was enhanced by preincubation with pertussis toxin (PTX), suggesting that Gs and inhibitory Gi/Go proteins mediate this response. Only activation of RXFP1 inhibited nuclear factor kappaB transcription, and this was reversed by PTX and the phosphoinositide-3-kinase inhibitor wortmannin. In addition, the glucocorticoid-response element was activated by RXFP1 but not by RXFP2 and was not activated in the parent HEK293T cells. Thus, the use of reporter genes enabled differences in signaling between these two receptors to be revealed and also threw light on the wide range of effects attributed to relaxin.