A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.