The definition of the genus Actinomyces relies heavily on traditional methods of taxonomy. This study sought to develop molecular tools for the identification of strains of Actinomyces israelii and Actinomyces gerencseriae. Oligonucleotide probes were designed and one of these successfully differentiated. A. gerencseriae from ten strains of A. israelii and three other Actinomyces species by DNA:DNA hybridization. However, probes based on known 16S rRNA sequences failed to hybridize to all the strains previously identified as A. israelii. Using the PCR technique, a region encoding a portion of the 16S rRNA was amplified from genomic DNA. The results showed that A. israelii can be divided into three different groups based on comparison of the amplified DNA sequences. This information should allow the development of probes that are specific for these newly identified groups of strains within the species A. israelii.