We have identified, employing two-dimensional (crossed) immunoelectrophoresis, a major heat-stable surface antigen in Actinomyces israelii with an electrophoretic mobility of 0.86 compared to bovine albumin. The surface component was protease sensitive and non-dialyzable and was found in relatively large amounts in two serotype 2 strains of A. israelii (WVU 307 and ATCC 23860). Serotype 1 strains either lacked antigen 0.86 (ATCC 12103 and CDC X522) or contained small quantities of a cross-reacting antigen which was not identical to antigen 0.86 (CDC W855). Thus, antigen 0.86 may be useful in the serotaxonomy of A. israelii. Our data also suggest the possibility that a greater degree of antigenic heterogeneity exists within the species than can be accommodated within the two currently recognized serotypes.