tRNA intergenic spacer PCR (tDNA-PCR) was evaluated for its usefulness in the differentiation of enterococcal species of human and animal origin. This technique was carried out for 124 strains belonging to 17 enterococcal species and generated DNA fragments, which were separated by capillary electrophoresis. tDNA-PCR enabled us to discriminate for all species tested. Enterococcus faecium showed minor but reproducible differences with Enterococcus durans, while Enterococcus hirae was easily distinguishable. Enterococcus avium, Enterococcus malodoratus, and Enterococcus raffinosus generated highly similar though distinctive patterns.