Based on previous success with rapidly growing mycobacteria, a microdilution MIC system was devised for slowly growing mycobacterial species using 7H9 broth. Test drugs included isoniazid, rifampin, ethambutol, streptomycin, clofazamine, and sulfamethoxazole. Sixty isolates of four mycobacterial species, including Mycobacterium tuberculosis, from patients who had never received drug therapy were evaluated in the system, as well as 25 drug-resistant isolates and 11 control strains. MICs were read when good macroscopic control growth was evident, a period which varied with each species. Most species exhibited a narrow range of MICs with easily discernible growth endpoints. The aminoglycosides, ethambutol, clofazamine, and sulfamethoxazole were the only drugs with activity against all species at clinically achievable levels in serum. Correlation between susceptibilities by the proportion method in agar with single drug concentrations and the broth method were excellent for M. tuberculosis, M. kansasii, and M. marinum for isoniazid, rifampin, and ethambutol. Isolates of the M. avium complex were much more susceptible in broth than in agar for rifampin, ethambutol, and streptomycin. Given the successful transition of most microbiology laboratories to MIC plates for other bacterial species, this method would allow for testing of multiple drugs at multiple concentrations and has good potential for evaluation of drug combinations and drug-resistant isolates.