Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.The genus Mycobacterium contains more than 100 species of nontuberculous mycobacteria (NTM) (28). Unlike the members of Mycobacterium tuberculosis complex and Mycobacterium leprae, NTM species are not obligated pathogens and are inhabitants of the environment. They can be found in natural water, water distribution systems, soil, protozoans, plants, and animals (24, 27). The emergence, in the last decade, of opportunistic NTM infections in humans has prompted the study of characteristics that allow mycobacteria to persist in their natural reservoirs. Motility has been related to the capacity of bacteria to colonize and persist in the environment. Mycobacteria were considered nonmotile microorganisms until 1999, when Martínez et al. (17) reported that Mycobacterium smegmatis and Mycobacterium avium spread on the surface of a growth medium by sliding motility. In these species, motility was related to colonial morphology and the presence of glycopeptidolipids (GPLs), a class of glycosylated peptidolipids located in the cell wall of some mycobacteria (for a recent review on GPLs, see the report of Chatterjee and Khoo [5]). Smooth colonies were motile and contained GPLs, but spontaneous rough colonies lacked GPLs and were nonmotile (17). Genetic evidence for the requirement of GPLs for sliding motility was later provided for M. smegmatis but not for M. avium (25). The latter...