When human chorionic gonadotropin (hCG) was treated with a 20-fold molar excess of N-acetylimidazole in aqueous solution, two tyrosyls were acetylated, resulting in a 50% reduction in in vivo biological activity. With increased quantities of reagent, the number of tyrosyls acetylated increased but with no further decrease in the biological activity. In vitro biological activity (binding to hCG receptors) of the hormone was not affected at all. Acetylation in urea increased tyrosyl modification, accompanied by acetylation of some lysyls, yielding a product in which all seven tyrosyls were acetylated and both in vivo and in in vitro biological activities were completely abolished. Deacylation with hydroxylamine partially restored biological activity of some but not all of the modified products. When individual hCG subunits were treated with the same reagent, the number of tyrosyls acetylated in each subunit again increased with increasing amounts of reagent, up to three in the alpha subunit and two in the beta subunit in the absence of urea. The tyrosyls in the beta subunit appeared less reactive to the reagent than those in the alpha subunit. Subunits modified to these extents retained ability to recombine as examined by gel electrophoresis, but the recombined products varied considerably in both in vivo and in vitro biological activities. A completely tyrosyl-acetylated product of hCG-alpha did not combine with intact hCG-beta, while fully modified hCG-beta did with intact hCG-alpha.