AbstractThe pathogenic fungus Candida albicans secretes a considerable number of hydrolases and other proteins. In‐depth studies of the C. albicans secretome could thus provide new candidates for diagnostic markers and vaccine development. We compared various growth conditions differing in pH, temperature and the presence of the hyphal inducer N‐acetylglucosamine. The polypeptide content of the growth media was ca. 0.1–0.2% of the total biomass. Using LC–tandem mass spectrometry, we identified 44 secretory proteins, the transmembrane protein Msb2, six secretory pathway‐associated proteins and 28 predicted cytosolic proteins. Many secretory proteins are wall‐related, suggesting that their presence in the growth medium is at least partially due to accidental release from the walls. Als3, Csa2, Rbt4, Sap4 and Sap6 were enriched in the medium of hyphal cultures; Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1, Scw11, Sim1/Sun42, Xog1 and Ywp1 in the medium of yeast cells; and Plb4.5 in pH 4 medium. Seven proteins (Cht3, Mp65, Orf19.5063/Coi1, Scw11, Sim1, Sun41 and Tos1) were consistently present under all conditions tested. These observations indicate that C. albicans tightly regulates its secretome. Mp65, Sun41, and Tos1 were each predicted to contain at least one highly immunogenic peptide. In total, we identified 29 highly immunogenic peptides originating from 18 proteins, including two members of the family of secreted aspartyl proteases. Fifty‐six peptides were identified as proteotypic and will be useful for quantification purposes. In summary, the number of identified secretory proteins in the growth medium has been substantially extended, and growth conditions strongly affect the composition of the secretome. Copyright © 2010 John Wiley & Sons, Ltd.