Immunoblot analysis showed that enolase is one of a subset of proteins found in cell supernatants of Candida albicans. Enzyme assays on whole cell extracts indicated that enolase is an abundant protein, comprising 0.7% and 2.0% of the total protein from yeast and hyphal forms of C. albicans, respectively. Comparison of enolase enzyme activities in whole cell extracts and cell culture supernatants showed the enzyme to be located primarily within cells. Extracellular glyceraldehyde-3-phosphate dehydrogenase activity was absent or lower than that of enolase, despite equivalent intracellular levels. The results suggest that enolase, released from fungi in the absence of host factors, may contribute to enolase found circulating in the blood of patients with hematogenously disseminated candidiasis. In addition, the release from cells of highly immunogenic fungal proteins, such as enolase, may be important in defining the selective stimulation of host antifungal responses during infection.