A method for preparing uniformly dispersed cultures of Nocardia asteroides for use in tests for susceptibility to antimicrobial agents is described. The minimal inhibiting concentration (MIC) of 45 agents for cultures thus prepared was determined with the use of a replica-inoculating apparatus. Minocycline at a concentration of 3.1 mug or less/ml inhibited 90% of the strains tested, and all were inhibited by 6.3 mug/ml. An erythromycin concentration of 0.8 mug or less/ml inhibited 40% of the strains, but the MIC for most of the others was > 100 mug/ml. The other agents were generally less active. Chemically related analogues varied in activity to different degrees. Also, the MIC of each antibiotic against different strains generally varied over a wide range. Sulfonamides and trimethoprim were not active against most strains in the method used. The size of the inoculum markedly affected the MIC of sulfonamides and had variable effects on other agents. Marked synergy of erythromycin with ampicillin was demonstrated for nearly all strains tested.