Toxicity and failure of treatment with amphotericin B are stimuli for researchers to evaluate alternative antifungal antimicrobics. Also, data from susceptibility tests of Coccidioides immitis are sparse. With use of a defined, synthetic culture medium, C. immitis (25 strains). Candida albicans (21 strains), and Cryptococcus neoformans (21 strains) were tested against flucytosine, clotrimazole, and amphotericin B. Molecule for molecule, the sequency of activity was: clotrimazole greater than amphotericin B greater than flucytosine (totally inactive) C. immitis; and clotrimazole greater than amphotericin B greater than flucytosine with C. albicans and C. neoformans. With four strains of C. immitis, the minimal inhibitory concentration (of amphotericin B) was the same when inocula of arthrospores were tested as when corresponding spherules/endospores were tested simultaneously and identically. The clinical outcome of coccidioidomycosis in 17 patients treated with amphotericin B correlated best with minimal inhibitory concentration after incubation of cultures for 48 hr; a favorable response was associated with minimal inhibitory concentrations of less than or equal 1.0 mug/ml. Because clinical isolates of fungi appear to vary in susceptibility, in vitro tests may have clinical utility.