Strains of Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum, Cryptococcus neoformans, Nocardia asteroides , and Coccidioides immitis were tested for in vitro susceptibility to polymyxin, gentamicin, kanamycin, chloramphenicol, and neomycin at concentrations of 1, 2, 4, 8, and 16 μg/ml. Polymyxin was the most inhibitory and gentamicin was the least inhibitory of the five antibiotics. Two Histoplasma mycelial strains were partially inhibited by 2 and 8 μg of gentamicin per ml and showed at least a 2+ growth at the higher antibiotic concentration. Kanamycin and neomycin produced significant inhibition of N. asteroides but otherwise were noninhibitory. A combination of chloramphenicol and kanamycin, each at 16 μg/ml, and gentamicin, at 4 μg/ml, was noninhibitory to the strains tested except for N. asteroides. Chloramphenicol at 16 μg/ml was not inhibitory for N. asteroides. The results suggest that the optimal antibiotic combination to use in the isolation of fungi and higher bacteria is chloramphenicol, 16 μg/ml, and gentamicin, 4 μg/ml. Addition of sheep blood (5%) had no effect on antibiotic susceptibility of the organisms studied.