Based on sequence homology to insulin and relaxin, we have isolated two novel genes of the insulin superfamily from mouse tissues. Because these proteins show a high similarity to relaxin and relaxin-like factor (RLF or Ley I-L), they were named as RIF1 (relaxin/insulin-like factor 1) and RIF2 (relaxin/insulin-like factor 2). After RT-PCR, full-length cDNAs of RIF1 and RIF2 were obtained from mouse testis and ovary, respectively. In addition, a putative human ortholog of RIF1 was isolated from human testis. The deduced coding regions of mRIF1, mRIF2, and hRIF1 were 191, 145, and 213 amino acids, respectively, and all three proteins contain a typical signal sequence for secretion at their amino terminus. Sequence comparison indicated that RIFs encode proteins consisting of B and A subunits connected by a long C domain peptide, and the deduced mature proteins of these putative ligands are most closely related to relaxin, RLF, and insulin from different species. Northern blot analysis showed that RIF1 transcripts are approximately 1.2 kb in size and are expressed mainly in testis of mouse and human. In contrast, RIF2 message of 2.0 and 1.2 kb are preferentially expressed in mouse kidney and are lower in testis, heart, and brain. In addition, immunohistochemical analysis showed that testis expression of RIF1 is restricted to interstitial cells surrounding seminiferous tubules. In kidney, the RIF2 message is localized to selected epithelial cells of loop of Henle. The exclusive expression pattern of RIF1 and related RLF in testis interstitial cells suggested potential physiological roles of these two distinct insulin/relaxin family ligands in testis function. Additionally, the spatial expression pattern of RIF2 suggests a novel role of RIF2 in nephrophysiology. Identification of RIF polypeptides expands the family of relaxin- and insulin-like hormones and allows future elucidation of the physiological role and hormonal mechanisms for these tissue-specific factors.