This paper reports the usefulness of two‐dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the identification of the major Candida albicans antigens. In order to have different patterns of protein expression and subcellular localization, three types of protein preparations were obtained: cytoplasmic extracts, protoplast lysates and proteins secreted by protoplasts regenerating their cell wall. These proteins were separated by high‐resolution two‐dimensional electrophoresis using an immobilized pH gradient. Western blotting with sera from patients with systemic candidiasis allowed the detection of more than 18 immunoreactive proteins. Some of these proteins had different isoforms. All sera reacted with at least three C. albicans proteins and the most reactive serum detected up to eleven proteins. Some of these antigens, e.g., enolase and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), have been identified on the 2‐D map. The most reactive proteins were enolase and a 34 kDa protein in the acidic part of the gel (pI 4—4.4) that was only detected in regenerating protoplast‐secreted proteins. The identification of all these antigens would be useful for the development of diagnostic strategies.