Mycobacterium simiae was the third most common mycobacterium identified over a 2-year period from a single clinical laboratory in southern Arizona. Thirty-three isolates from 25 patients were identified over 1 year. The isolation of M. simiae was considered clinically significant for only two of 23 evaluable patients. None of five patients with human immunodeficiency virus infection had clinical disease associated with M. simiae. Twenty isolates were available for detailed study. All but one of the 20 isolates were niacin-negative, and 11 were nonphotochromogenic. All 20 isolates had a triple-cluster pattern consistent with M. simiae by high-performance liquid chromatography, and restriction fragment patterns were identical for 16 isolates. Analysis of 16S rDNA confirmed the identity of all the tested isolates as M. simiae. In this study, M. simiae was a frequent clinical isolate but was rarely associated with disease. The organisms isolated were confirmed to be M. simiae but appeared to be phenotypically distinct strains of low virulence.