To determine the serum concentrations of human chorionic gonadotropin (hCG), its free beta-subunit (hCG beta), and the free alpha-subunit (free alpha) common to all human glycoprotein hormones under physiological and pathological conditions, we developed monoclonal antibody-based immunoenzymometric assays. Free alpha-subunit was detected in the sera of all healthy individuals of both sexes; hCG was measurable in sera of 54% of the men, and 46% were positive for free hCG beta; in nonpregnant women, 69.5% were positive for hCG, 68.4% for the free beta-subunit. Pathological conditions, i.e., hCG-producing tumors, were studied in vitro and in vivo. In vitro, the concentrations of hCG, free hCG beta, and free alpha in tissue-culture supernates of a choriocarcinoma cell-line ("JAR") showed a parallel pattern during time-course analysis. In vivo, in long-term follow-up studies of 13 patients with testicular cancer, serum concentrations of the three analytes paralleled each other, whether the disease was in remission or not. Because of a selective increase of free hCG beta and free alpha in 27% of seminomatous tumor patients and in 13% of the nonseminomatous patients, the percentage of tumor-marker-positive sera was increased from 15% to 42% and 57% to 70%, respectively, by the additional measurement of free hCG beta and free alpha. Thus hCG, free hCG beta, and free alpha are physiologically present in a high percentage of the sera from healthy men, and the determination of free hCG beta and free alpha, although not of prognostic value, improves the diagnostic possibilities in patients with testicular cancer.