We investigated the effects of various test conditions on broth macro- and microdilution susceptibility test results for several species of yeasts with a new antifungal agent, cilofungin. As the pH decreased from 7.4 to 3.0, 50% inhibitory concentrations (IC1/2) increased up to 64-fold. This effect was unrelated to yeast growth rate, solvent concentration, or choice of buffer. Broth microdilution results for 42 Candida albicans isolates at pH 7.4 in synthetic amino acid medium, fungal (SAAMF), showed IC1/2 results from 0.08 to 2.5 micrograms/ml, whereas at pH 3.0 the results were 5.0 or 10.0 micrograms/ml. Fungicidal concentrations were closer to MIC results at the lower pH, i.e., an average of 16-fold above the MIC at pH 3.0, compared with an average 256-fold difference at pH 7.4. Two strains that had very different IC1/2 results at pH 7.4 and identical IC1/2 results at pH 3.0 were found to be equally susceptible to cilofungin therapy in rats. In additional studies, other medium effects were demonstrable, with yeast nitrogen broth and minimal essential medium generally yielding higher results than two other synthetic media (SAAMF and RPMI 1640 medium). However, susceptibility results did not change with inoculum between 10(2) and 10(5) yeast cells per ml or temperature between 30 and 37 degrees C. These studies indicate that pH is an important influence on in vitro rank order susceptibility of pathogenic yeasts to cilofungin.