Rat relaxin, as isolated from ovaries, has been described in the literature as a low potency hormone in the mouse symphysis pubis assay. Searching for an explanation, a helix-breaking glycine residue in the B chain seemed to be the most auspicious perturbation. Rat relaxin was chemically synthesized and analyzed by reverse-phase high performance liquid chromatography, amino acid composition, mass spectrometry and circular dichroic spectroscopy. Analogs of rat relaxin were synthesized either with aspartic acid in place of the helix-breaking glycine residue in the receptor-binding region of the B chain or with Asp-Leu-Val instead of Gly-Tyr-Val at positions B14-B16. In receptor-binding assays [B14D, B15L, B16V]relaxin was a better ligand than rat relaxin, whereas the [B14D]relaxin was less potent. In the mouse symphysis pubis assay, both analogs were less potent than unmodified rat relaxin, but the [B14D, B15L, B16V]relaxin was better than [B14D]relaxin. In contrast to previous reports on native rat relaxin, the chemically synthesized rat relaxin proved to be as active as human and porcine relaxin with respect to the standard mouse assay system. Glycine, which is considered to be a perturbator in an alpha helix, is not only tolerated in the B14 position but is required for full biological potency.