Mycobacterium haemophilum, first described in 1978, can cause severe infections of skin, respiratory tract, bone, and other organs of immunocompromised patients. There is no standardized antimicrobial susceptibility test, and for the 27 reported cases, a variety of test methods have been used. This paper reports the in vitro test results for 17 isolates of M. haemophilum recovered from 12 patients in the New York City area. MICs of 16 antimicrobial agents were determined in microtiter trays containing Middlebrook 7H9 broth plus 60 ,uM hemin, inoculated with 106 CFU of the organism per ml and incubated at 30'C for 10 days. Ethambutol, ethionamide, tetracycline, cefoxitin, and trimethoprim-sulfamethoxazole were inactive against initial isolates from the 12 patients. Isoniazid was weakly active with a MIC for 50%o of strains tested (MIC50) of 8 ,ig/ml and a MIC90 of >32 ,ug/ml. Three quinolones, ciprofloxacin, ofloxacin, and sparfloxacin, were moderately active with MIC50s of 2 to 4 ,ug/ml and MIC90s of 4 to 8 ig/ml. Amikacin and clofazamine were active with MIC90s of 4 and 2 ,Ig/ml, respectively. Clarithromycin was the most active macrolide with a MIC90 of .0.25 pg/mI. The MIC90 of azithromycin was 8 pg/ml, and the MIC90 of erythromycin was 4 ,ug/ml. The rifamycins were active with a MIC90 of 1 ,ug/ml for rifampin and one of <0.03 pg/ml for rifabutin. For a second isolate from the skin of one patient and an isolate from an autopsy culture of the spleen of a second patient, MICs of rifampin and rifabutin were > 16 p,g/ml, whereas initial isolates were inactivated by low concentrations of the rifamycins. Both patients had been treated for several months with several antimicrobial agents, including a rifamycin.