Antigenic relatedness among human strains of oral Actinomyces and similar isolates from cattle has been analyzed by agglutination and immunoblotting. Whole cell agglutination placed A. viscosus serotype II, A. naeslundii serotypes II and III, Actinomyces NV, and strains from numerical taxomonic clusters C1, C2, C3, C4, and C6 into a single group. A. viscosus serotype I cross-reacted weakly with this group. A naeslundii serotype I strains and the cattle isolates Actinomyces denticolens and Actinomyces howellii were distinct. The agglutination results for A. slackii were equivocal. Immunoblots of cell wall extracts developed with non-absorbed sera showed cross-reactivity (23% to 90% antigenic similarity) among all of the strains tested, including A. israelii. The range of antigenic similarities among the group which included strains of A. viscosus serotype II, the A. naeslundii serotypes, and clusters C1, C2, C3, C4, and C6 was from 39% to 89%. Immunoblotting showed that A. howellii and A. denticolens were between 39% and 72% similar to A. naeslundii and A. viscosus. Absorption of antisera with A. israelii cell walls removed antibodies recognizing antigens common to Actinomyces and made the sera more specific. Immunoblotting with absorbed sera supported the grouping and separation of strains shown by agglutination. In some cases, serotypes could be included into a specific taxonomic cluster. A. naeslundii serotype II and Actinomyces NV most closely resembled cluster C1 strains, and A. naeslundii serotype III resembled cluster C1 strains, and A. naeslundii serotype I and A. viscosus serotype I were included into clusters C5 and C7, respectively. The results support a recent proposal that strains of A. viscosus serotype II, A. naeslundii serotypes II and III, and Actinomyces NV be included into A. naeslundii genospecies 2, that A. naeslundii serotype I should be designated A. naeslundii genospecies 1, and that A. viscosus serotype I should be retained distinct from A. naeslundii, as A. viscosus.