A sensitive, accurate, and reproducible in vitro bioassay was developed for measuring human chorionic gonadotropin (hCG), based on testosterone production by collagenase-dispersed interstitial cells of rat testes in response to hCG. The results were compared to those obtained by established beta hCG radioimmunoassay. The assay sensitivity was 25 pg hCG-CR119/ml (65 microIU second IRP/ml). The intra-assay coefficient of variation (CV) was 8.8%, and the interassay CV was 13% and 33% in the high and low ranges of the standard curve, respectively. hCG recoveries were 89.6 +/- 3.12% (SE, n = 12). The pattern of serum bio-hCG followed established patterns of immuno-hCG, with the highest level measured during the first trimester (mean, 52,600 +/- 7,250 SE (mIU/ml, n = 11), decreasing thereafter to a mean value of 7,400 +/- 1,500 mIU/ml at term. The mean ratio of the bio/immunoactivity was consistently greater than one and did not significantly change at the various stages of pregnancy, or between normal and molar pregnancies (first trimester, 1.75 +/- 0.12 SE; midtrimester, 1.46 +/- 0.12; term, 1.50 +/- 0.09; molar, 1.55 +/- 0.2). When serum bioactive and immunoactive hCG were measured in a woman at five weeks of pregnancy, an episodic secretion of hCG was obtained by both assays.