A zymogram method, following thin-layer isoelectric focusing in a polyacrylamide gel, allows resolution of the lytic activity of serum C2 complement protein in a spectrum of molecular forms. This spectrum is characteristic in each of the species studied (man, rhesus monkey, guinea pig, and hamster). Moreover, two different alternative patterns are observed in man: each of the six major lytic bands characteristic of the most common pattern (herein designated C21) is duplicated in the least common pattern (C21), with an additional band displaced cathodally by not more than 0.04 pH unit. Distribution of phenotypes C21 and C2(2-1) in a Caucasion population is in agreement with the hypothesis that they are controlled by two alleles, C21 and C22, with frequencies 0.96 and 0.04 ± 0.01. Segregation studies show that the two alleles are codominant and identify a locus in the HLA region. No recombinants with HLA-B were detected among 27 informative meioses, generating a cumulative lod score of 6.321 at = 0. These findings suggest that the individuals with the C2 deficient trait might be interpreted as homozygotes for a third and rarest amorph C20 of the same locus.