Current agar dilution methods for susceptibility testing of Mycobacterium marinum versus the tetracyclines have failed to show in vitro susceptibility despite good in vivo results. We found that the tetracyclines were unstable in agar and resulted in a fine haze of growth for several concentrations before complete inhibition of growth was seen. In contrast, the aminoglycosides resulted in sharp minimal inhibitory concentration endpoints, which were generally independent of the length of incubation. The problems with the tetracyclines can be lessened by shortening the incubation time to 5 days and redefining the minimal inhibitory concentration as the lowest concentration resulting in no growth or a slight haze of growth. By this methodology, 19 clinical isolates of M. marinum were tested for susceptibility to the tetracyclines and aminoglycosides. Minocycline inhibited greater than 50% of isolates at 2.0 microgram/ml, whereas both minocycline and doxycycline inhibited greater than 90% of isolates at 4.0 microgram/ml. These studies support the usage of the tetracyclines in the treatment of clinical diseases due to M. marinum and suggest a susceptibility method which is more predictive of clinical results.