We have investigated the application of a RIA for porcine relaxin to the assay of relaxinlike substances in the blood of various other mammalian species. The cross-reactivity between antiporcine relaxin antibody and the relaxinlike substances in the blood of other mammals during pregnancy was sufficiently high to permit the assay of 0.1-0.5 ml of serum or plasma samples. Nonspecific reactivity was controlled by adding similar volumes of serum or plasma obtained from ovariectomized female or intact male subjects to RIA tubes containing known porcine standards. RIA relaxin levels rose markedly during the last third of pregnancy in rats, mice, and guinea pigs. RIA relaxin was also found in late pregnancy in dog, rhesus and java monkeys, and human beings. The apparent blood levels of hormone found in each species will depend upon their degree of cross-reactivity with the antiporcine relaxin antibody as well as upon their actual concentration. Thus, absolute blood level values should not be taken literally. However, the fluctuations in RIA relaxin observed during the course of gestation in any given species would appear to reflect accurately the relative blood concentrations of the hormone.