An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluoresceinlabelled PCR product was partially digested with restriction site enzymes BstEII and HaelII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of __1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffinembedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. boris, M. boris bacille Calmette-Gudrin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enzyme sites.