In a study to determine if relaxin could be measured in milk and if so to correlate concentrations in milk and serum, paired samples of milk and serum were collected from 12 women 3 days after term delivery (term group), from 16 women 3 days after preterm delivery (preterm group), and from some of these patients 6 weeks after delivery (eight term and six preterm). Relaxin was measured by specific human relaxin radioimmunoassay. Relaxin from milk and sera behaved similarly in the relaxin radioimmunoassay and reverse-phase high-performance liquid chromatography. Concentrations of relaxin in sera and milk collected 3 days after delivery did not differ significantly within the term or preterm groups. Neither were there differences in relaxin levels in sera and milk between the term and preterm groups. At 6 weeks postpartum, relaxin was not measured in any sera but was measured in milk from six of eight patients in the term group and five of six patients in the preterm group. Relaxin concentrations in milk were higher in the preterm group. The presence of relaxin in milk at 6 weeks postpartum suggests a nonluteal site of synthesis.