Peptide variations in the alpha-subunit (molecules starting at alpha 3 and alpha 4) and beta-subunit (missing linkages at beta 44-45 and beta 47-48) of hCG have been reported by several investigators. Studies, however, have been limited to standard hCG preparations (purified from large pools of urine) and other hCG samples from mixed urines. In this study we used chromatographic procedures to purify the total hCG content of 13 individual urines, 6 from patients with pregnancy and 7 from those with trophoblast disease (no hCG-containing fractions were excluded). Then, we examined for the first time the peptide variability among individual samples of hCG. We report 1) that individual hCG preparations have nicks (missing linkages) in the beta-subunit, primarily between residue 47-48 (11 of 13 samples) and, less commonly, at the linkage 44-45 or 46-47 (3 of 13 samples); 2) the extent of nicking varies greatly between individual preparations (range, 0-100% of molecules); 3) varying alpha-subunit N-terminal heterogeneity (N-terminus starting at alpha 3 or alpha 4) was also present (range, 0-28% of molecules), but was confined to preparations from individuals with trophoblast disease (6 of 7 samples from trophoblast disease urine, 0 of 6 from pregnancy urine); 4) hCG missing the beta-subunit C-terminal region was also detected (2 of 13 hCG preparations); and 5) 1 of 13 preparations was nicked on the hCG alpha-subunit, between residues 70 and 71. Thus, 12 of 13 individual hCG samples demonstrated at least 1 of 4 different forms of peptide heterogeneity. We conclude that individual hCG samples vary widely in the type and extent of peptide heterogeneity, an observation that is not appreciated when pools of hCG are studied.