To determine whether the hCG-like substance found in the plasma of normal nonpregnant subjects is secreted and regulated like a pituitary gonadotropic hormone or prohormone, we measured its concentration before and 60 min after the acute iv injection of 500 microgram Gonadotropin-releasing hormone (GnRH). The study was performed in 12 normal young men. The hCG-like substance was purified from the other plasma proteins by 2 successive diethylaminoethyl-Sephadex A-50 chromatographies and by gel filtration on Sephadex G-100. Its concentration was measured by a specific RIA, corrected for losses and for possible contamination with human LH (hLH). The median basal concentration in plasma was 19 pg/ml (mean, 41; range less than 5 to 169), and there was no correlation with corresponding hLH concentrations. After the injection of GnRH, the median, mean, and range of concentrations remained constant, whereas the mean hLH concentration increased over 8-fold. This indicates that no significant amounts of the hCG-like substance are acutely releasable from the pituitary gland and that its plasma concentrations are not modulated by GnRH in a fashion similar to hLH. It also constitutes an argument for an extrapituitary origin of the plasma hCG-like substances in normal men.