We have isolated rat relaxin as a single chromatographic component and determined its complete amino acid sequence. Comparison with porcine relaxin reveals a clear sequence homology but the extent of this is much more limited than expected, with only 21 of 53 residues (39.6%) in corresponding positions being identical. In contrast 47 of 51 residues (92.2%) are identical between porcine and rat insulins. These findings suggest that relaxin has been less constrained to conserve its structural features during evolution than has insulin, since the original duplication of a common ancestral gene. Relaxins as a group (and rat and porcine relaxins in particular) show poor immunological cross-reactivity despite a common biological activity. The finding of extensive sequence differences as well as conserved residues between porcine and rat relaxins provides a possible structural basis for these observations, and should facilitate identification of a localized receptor-binding surface region of relaxin. A further important implication of this limited sequence homology is that physiological (and clinical) studies with relaxins should whenever possible be carried out with hormone preparations from the same species.