A molecular assay that distinguishes among strains of the periodontal pathogen Actinobacillus actinomycetemcomitans was developed by identifying DNA restriction site polymorphisms in the highly variable transcribed spacer region between the 16S and 23S ribosomal genes. The polymerase chain reaction (PCR) was used to amplify this region from genomic DNA using primers within conserved regions of the 16S and 23S genes. This amplified region was digested using a series of restriction enzymes and electrophoresed. Examination of restriction fragment length polymorphisms obtained by separate digestion with RsaI and NciI allowed the 7 strains examined to be divided into 4 genetic groups. This assay provides a more precise and reproducible method of strain identification than whole genomic methods and should be useful as a method for studying the epidemiology of A. actinomycetemcomitans strains in human subjects. The genetic variability detected supplies strong evidence that direct sequence analysis of the region could provide extremely precise and potentially definitive identification of strains.