Immunocytochemical methods were used for the demonstration of Actinomyces israelii, Actinomyces naeslundii and Arachnia propionica in bacterial colonies found in 7 routine biopsies from periapical inflammatory lesions. All 3 species were found in one specimen, both A. israelii and A. propionica in 3 specimens, and one of each species in the remaining 3 biopsies. Specificity controls by enzyme immunoassay showed that antiserum to A. israelii reacted also with A. odontolylicus, and that to A. naeslundii with A. viscosus, while antiserum to A. propionica did not show any cross‐reactions with Actinomyces species. The results indicate that immunocytochemical methods can be used for the diagnosis of periapical actinomycosis at the species level. Actinomyces and Arachnia species seem to have an important role in the pathogenesis of complicated periapical infections.