The in vitro susceptibility of three strains of an unclassified Mycobacterium sp., isolated from three patients with Crohn's disease, to 23 antimicrobial agents was determined by a modified broth dilution method with 7H9 broth containing oleic acid-albumin-dextrose-catalase, Tween 80, and mycobactin J. All three strains were susceptible to streptomycin, viomycin, rifampin, clofazimine, cefazolin, amikacin, and kanamycin and resistant to p-aminosalicylic acid, cycloserine, 2-thiophenecarboxylic acid hydrazide, trimethoprim, diaminodiphenylsulfone, sulfamethoxazole, sulfadimethoxine, polymyxin B, metronidazole, neomycin, and carbenicillin. Variable results between strains were encountered with ethambutol, ethionamide, capreomycin, amoxicillin, and cephalothin.