A newly developed, specific, sensitive and reliable RIA for rat relaxin is described. Radioiodinated rat relaxin, with a specific activity of 22 to 37 kBq/micrograms, was prepared from purified authentic ovarian rat relaxin using the chloramine T method. Polyclonal rat relaxin antiserum was raised in rabbits. Assay sensitivity was 30 pg/tube. Intra- and interassay coefficients of variation were 8% and 13%, respectively. Initial experiments demonstrated rising concentrations of rat relaxin in pregnant rats. Serum of adult male or non-pregnant female rats contained undetectable levels of rat relaxin. The assay is suitable for measurements of rat relaxin in biological fluids, thus making the assay a valuable tool for further studies on the physiology of relaxin.