The purpose of this study was to determine whether or not the immunoreactive relaxin contained in human pregnancy serum is biologically active. Relaxin was extracted from pooled serum of women in the third trimester of pregnancy. The extraction also was performed on pooled male serum and pooled serum from women in the follicular phase of the menstrual cycle. The extraction process began with delipidation of the serum followed by extraction of relaxin using 0.1% HCI per 50% acetone. Dialysis then was performed to remove low molecular weight substances (less than 3500 daltons). The partially purified extracts were applied individually to a muscle segment in the in vitro rat uterine horn bioassay for relaxin. The extracts of both male serum and female follicular-phase serum, containing no immunoreactive relaxin, resulted in tetanic contractions of the muscle segment. The extract of pregnancy serum, containing immunoreactive relaxin, produced inhibition of rat uterine horn contractility. This represents the first demonstration of relaxinlike bioactivity in human serum.