Ketoconazole was tested in vitro in three different media against 69 isolates of pathogenic fungi by using a macro-broth dilution procedure. The dimorphic systemic pathogens were highly susceptible, with most isolates of Blastomyces dermatitidis and Histoplasma capsulatum being inhibited and killed by concentrations less than or equal to 0.39 micrograms of ketoconazole/ml. Most isolates of Coccidioides immitis were also inhibited or killed by 0.39 micrograms of ketoconazole/ml; however, several were not killed by 100 micrograms/ml. Isolates of Cryptococcus neoformans and Sporothrix schenckii appeared to be less susceptible, with many isolates being resistant to less than or equal to 1.56 micrograms of ketoconazole/ml. There were 19 isolates of B. dermatitidis, C. immitis, and H. capsulatum recovered from 12 patients either during or following treatment with ketoconazole. Evidence for selection of secondary resistance to ketoconazole in these isolates was not observed. Results of these in vitro studies correlated poorly with the clinical responses to ketoconazole observed in the patients from whom the isolates were recovered.