Mycobacterium xenopi and Mycobacterium avium complex (MAC) are biochemically similar. To define the laboratory characteristics of M. xenopi that distinguish it from MAC, 53 M. xenopi isolates from different areas in the United States and 47 isolates recovered at one hospital were evaluated by 13 biochemical tests, AccuProbe MAC (Gen-Probe, Inc., San Diego, CA, USA), colony morphology, formation of X-colonies, pigmentation in response to light, growth on MacConkey agar without crystal violet, and relative growth rates at 25 degrees C, 36 degrees C, and 45 degrees C on solid media. Relative growth rates of 10 M. xenopi and 11 MAC isolates were measured at 25 degrees C, 36 degrees C, and 42 degrees C in Middlebrook broth processed using the BACTEC TB System. Ten M. xenopi were tested for p-nitro-alpha-acetylamino-beta-hydroxypropiophenone inhibition at 36 degrees C and 42 degrees C. Reevaluation of 81 isolates previously identified as MAC by biochemical tests alone revealed that two were M. xenopi. The most reliable characteristics distinguishing M. xenopi from MAC were the presence of X-colonies (M. xenopi 97% vs MAC 1%), positive 3-day arylsulfatase (M. xenopi 88% vs MAC 1%), growth at 25 degrees C (M. xenopi 0% vs MAC 100%), and AccuProbe MAC test results (M. xenopi 0% hybridized). Retrospective chart review of 37 patients using American Thoracic Society criteria revealed that six (16%) patients had clinically important isolates. At one of our hospitals M. xenopi was the second most common mycobacterial species isolated for 1990-1992, accounting for 27% of all isolates, whereas at our other hospital it accounted for 1% of isolates.(ABSTRACT TRUNCATED AT 250 WORDS)