To further characterize the hCG-like substance found in normal nonpregnant human tissues, we compared the biological (B) and immunological (I) activities of the substance in extracts of 40 nontrophoblastic tissues and 7 placentas. B was measured in an in vitro mouse testicular interstitial cell bioassay, and I was measured in the beta hCG RIA. Highly purified hCG (CR 119) was used as a standard. B was detected in 92% of the nontrophoblastic nonpituitary tissues. Parallelism between serial dilutions of extracts and the hCG standard was found for 75% of the pituitary extracts, 86% of the placental extracts, and 53% of the other tissues. The correlation between B and I activities in the nontrophoblastic nonpituitary tissues was good (r = 0.8; P less than 0.001). Preincubation of the tissue extracts with anti-hCG serum before bioassay significantly reduced the B activity. There was wide variation in the B to I ratio between tissues and within the same tissues from different individuals. A B to I ratio less than 1 was found in 50% of the tissues, excluding placenta and pituitary, and may have been due to the presence of incomplete or altered hCG molecules, which are immunologically active but have reduced B activity.