Saccharomyces cerevisiae was isolated in large numbers from operative specimens from two patients with perforated bowel and peritonitis and from the blood of another patient treated with extracorporeal membrane oxygenation. Susceptibility studies were performed on these three isolates and another 29 isolates that colonized or caused infection in a total of 19 patients seen over the last decade. All isolates had low minimum inhibitory concentration (MIC) values for amphotericin B (MIC90 of < or = 0.02 microgram/ml) and flucytosine (MIC90 of 0.2 microgram/ml), and a broader range of MIC values for itraconazole (MIC90 of 0.8 microgram/ml) and fluconazole (MIC90 of 4 micrograms/ml). A colorimetric method using Alamar blue reagent showed good concordance with the standard broth macrodilution method for amphotericin B, flucytosine, and fluconazole, but less good concordance for itraconazole. Serious infections with S. cerevisiae probably should be treated with amphotericin B, with or without the addition of flucytosine.