A distinct group of slowly growing mycobacteria was identified on the basis of growth characteristics, biochemical and lipid profiles, and nucleic acid analyses. The isolates showed growth at 22 to 37 degrees C, yellow pigmentation, and negative tests for Tween 80 hydrolysis, nicotinic acid, nitrate reductase, and urease; tests for arylsulfatase, pyrazinamidase, and heat-stable catalase were variable. Analysis of cellular fatty acids by gas-liquid chromatography and mycolic acids by thin-layer chromatography and high-performance liquid chromatography indicated a distinctive pattern which was unlike those of other species. Determination of the 16S rRNA gene sequence showed a unique sequence closely related to Mycobacterium simiae and M. genavense. On the basis of DNA homology studies, we suggest that these organisms are representatives of a novel species, for which the name M. lentiflavum sp. nov. is proposed.