Aims: To test the hypothesis that strains of Candida dubliniensis and C. albicans can be differentiated on the basis of polar lipid profiles. Methods and Results: Five isolates of C. dubliniensis and six isolates of C. albicans were tested by growth at 45 degreesC, production of chlamydospores on cornmeal agar, colonial colour on CHROMagar Candida medium and assimilation of DL-lactate, alpha-methyl-D-glucoside and xylose. Polar lipids were then extracted from freeze-dried cultures and analysed using fast atom bombardment mass spectrometry. Isolates were grouped by single linkage clustering based on correlation coefficients for strain pairs calculated with carboxylate and phospholipid molecular species distributions. The most intense carboxylate and phospholipid molecular species anions were of m/z 281 (C-18:1) and m/z 515 (PA 23 : 2). Phosphatidylethanolamine and phosphatidylglycerol were the predominant phospholipid families in C. dubliniensis, compared with phosphatidic acid in C. albicans isolates. All of the C. dubliniensis isolates grouped together in one cluster, whereas all of the C. albicans isolates grouped in a separate cluster. Conclusions: Fast atom bombardment mass spectrometry can differentiate the two species based on analysis of polar lipid distributions. Significance and Impact of the Study: These findings demonstrate that C. dubliniensis and C. albicans have distinct polar lipid profiles.