Prototype strains of human adenovirus (AV) (serotypes 1-35) as well as 49 wild strains were studied for reactivity of early antigens (EAs) with type-specific rabbit immune sera, using the immunoperoxidase antibody technique for determination of virus-specific IgG. The EAs of all AV serotypes belonging to one group reacted strongly with all type-specific immune sera of the homologous group. Reactivity with antisera to heterologous groups was moderate or absent except for the EAs of AV type 4, which reacted strongly with all AV-immune sera. Thus, on the basis of EA reactivity, all AV strains appeared to fall into five distinct groups (corresponding to DNA homology groups): group A, types 12, 18, and 31; group B, types 3, 7, 11, 14, 16, 21, 34, and 35; group C, types 1, 2, 5, and 6; group D, types 8-10, 13, 15, 17, 19, 20, 22-30, 32, and 33; and group E, type 4. For diagnostic virology, EA grouping is a valid alternative to differential hemagglutination; it is more complete, and all AV strains can be rapidly classified.