Mycobacterium fortuitum and Mycobacterium chelonei are distinguished unambiguously by the combined use of five test characters: nitrate reductase, beta-glucosidase, acid production from fructose, penicillinase, and trehalase. Typically, M. fortuitum was nitrate reductase positive, beta-glucosidase positive; M. chelonei was nitrate reductase negative, beta-glucosidase negative, penicillinase positive, and trehalase positive and did not produce acid from fructose.