The esat-6 and cfp-10 genes are essential for virulence in Mycobacterium tuberculosis. Among nontuberculous mycobacteria, we found these genes only in M. kansasii, M. szulgai, M. marinum, and M. riyadhense, with unique sequences. This adds a phylogenetic and taxonomical characteristic and may represent a virulence factor for nontuberculous mycobacteria.The 6-kDa early secretory antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP-10) of Mycobacterium tuberculosis are potent T-cell antigens (2, 12). The genes encoding ESAT-6 and CFP-10 are situated within "region of difference 1" (RD1) of Mycobacterium tuberculosis but are also present in some nontuberculous mycobacteria (NTM) (2, 12). Deletion of RD1 in M. tuberculosis significantly decreases its virulence in animal models (8), suggesting that genes residing in RD1 are involved in pathogenesis. In M. tuberculosis, the RD1 proteins effect translocation to the cytosol, a mechanism for survival within macrophages (15). Therefore, RD1 in NTM may also play a crucial role in virulence.To improve our understanding of the pathogenesis of NTM disease and the possible role of RD1 in virulence, we screened a wide diversity of NTM for the presence of RD1.From our laboratory database, we retrieved isolates of all five Mycobacterium kansasii subtypes based on 16S-23S internal transcribed spacer sequencing (n ϭ 15),M. riyadhense (n ϭ 1), and M. tuberculosis H37Rv.The selection of these strains was based on their phylogenetic relationship with the M. tuberculosis complex in the multigene taxonomical model published by Devulder et al. (3).To establish the presence of an RD1-like element and sequences of the esat-6 and cfp-10 genes, we used the Esa-12 (CATGACAGAGCAGCAGTG) and Esa-303 (5Ј-GCCCT ATGCGAACATCCC-3Ј) primers for esat-6 and the opBR78 (5Ј-GTAGCCCGGGATGGCAGAGATGAAGACCGATGC C-3Ј) and opBR103 (5Ј-TCAGAAGCCCATTTGCGAGGAC AGC-3Ј) primers for cfp-10 (1). The M. smegmatis esat-6 and cfp-10 gene sequences were extracted from the whole-genome sequence in the GenBank database (accession number CP000480).Using these primers, we were able to demonstrate an RD1 (1). None of the PCRnegative species hybridized with either probe (data not shown). The presence of RD1, characterized by an esat-6-like gene and an cfp-10-like gene, is a phylogenetic characteristic among the NTM. It is mainly found only among slowly growing NTM species that are phylogenetically related to the M. tuberculosis complex based on the multigene taxonomical model by Devulder et al. (3) and in the more distantly related rapid grower M. smegmatis. Possibly, the presence of RD1 reflects phylogenetic relationships to the M. tuberculosis complex.Previous authors have recorded the presence of RD1 in M. flavescens (1). We were unable to demonstrate it in four reference strains (ATCC 23008, ATCC 23033, ATCC 23035, and ATCC 23039) and a clinical isolate. Thus, with its presence of an RD1 region, M. smegmatis still stands out among the rapid growing NTM.Gey van Pittius et al. have demonstrated the presence ...