Rhodococcus species are ubiquitous in the environment, and several species have been reported to have pathogenic potential for humans. Rhodococcus equi, in particular, has been reported to cause infections in patients with AIDS. However, the identification of Rhodococcus species with use of conventional biochemical tests is problematic, and no simple and reproducible method exists for their rapid identification and differentiation. We found that the type strains of the 20 recognized species in the genus Rhodococcus could be clearly distinguished with use of a combination of the Pvu II and Pst I rRNA gene restriction endonuclease patterns and a digoxigenin-labeled Escherichia coli rDNA probe. Analysis of four clinical or environmental isolates confirmed as Rhodococcus bronchialis showed no interstrain variation of rRNA gene bands. Analysis of 15 isolates confirmed as R. equi from 13 patients showed 11 different rRNA gene patterns. No discernible difference was observed in the ribotype patterns between R. equi isolates from patients for whom AIDS had been diagnosed and those from patients who did not have AIDS, and there was no evidence of geographic clustering of R. equi ribotype patterns. Three of five Rhodococcus species isolates that could not be differentiated with use of conventional biochemical methods were identified with use of ribotype analysis. Therefore, ribotype analysis may provide an important adjunct to current biochemical identification of environmental and clinical isolates of Rhodococcus species.