A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.Mycobacteria other than Mycobacterium tuberculosis (MOTT) are increasingly recognized as causing human infections (31). Conventional biochemical methods and phenotypic tests for species differentiation are laborious and time-consuming and frequently require specialized testing that is beyond the capacity of clinical laboratories. Genotypic methods for the identification of mycobacteria have been developed in recent years (3,10,23,29). These molecular methods are gaining increasing importance because they yield rapid and, in most cases, unequivocal results.In 1992 Plikaytis et al. (15) developed a method for differentiating among slowly growing Mycobacterium species by PCR and restriction fragment length polymorphism analysis (PRA). A similar approach was used by Telenti et al. (27) for rapid identification of mycobacteria to species level based on evaluation of the gene coding for the 65-kDa heat shock protein (22) by PCR and restriction enzyme analysis. Subsequently, this approach was used for the taxonomic separation of rapidly growing mycobacteria (18, 24), for routine identification of mycobacteria (1,4,7,9,11,12,23,26), and for identifying Mycobacterium leprae (16,25) and Mycobacterium kansasii subspecies (17).In all these studies the algorithm describing the mycobacteria species is based on the use of two restriction enzymes (BstEII and HaeIII) and separation of the restriction fragments on an agarose gel. PRA patterns are then interpreted by converting the running distance in electrophoresis to apparent molecular size (in base pairs). Difficulties in PRA interpretation may stem from similarities in a number of band sizes that are critical for discrimination of species and are not sufficiently resolved by agarose-based gel electrophoresis.In view of the application of PRA-based identification of mycobacteria in our diagnostic laboratory and its application to an increasing number of different species, we conducted the present study in order to propose an algorithm based on 10% polyacrylamide gel electrophoresis (PAGE) of restriction digests to improve the resolu...