The peptide hormone relaxin was isolated in good yield from the ovaries of the pregnant rodent Rattus rattus using a simplified purification schedule. It was subjected to comprehensive chemical characterization to confirm both its purity and predicted composition. The peptide was also chemically synthesized by the solid phase procedure. The two chains comprising the hormone were each assembled by the Boc-polystyrene method and, following conventional purification, combined in solution to form the single intramolecular and two intermolecular disulfide bonds. Following purification, the synthetic rat relaxin was fully chemically characterized and shown to be indistinguishable from the native peptide including by secondary structure analysis using circular dichroism spectroscopy. The native and synthetic rat relaxins were shown to be equally biologically active in the in vitro rat uterine relaxation assay and had pEC50 values that were comparable to synthetic human H2 relaxin.