The mycelial surface antigen recognized by monoclonal antibody (mAb) 4E1 has previously been shown to be present predominantly in the terminal third of the hyphal structures in Candida albicans. We report here the expression cloning of the corresponding gene (CSA1 ) by mAb 4E1-coated magnetic beads sorting of Saccharomyces cerevisiae transformants expressing a C. albicans genomic library. The strategy is both highly selective and highly sensitive and provides an additional genetic tool for the cloning and characterization of C. albicans genes encoding surface proteins. CSA1 is an intronless gene encoding a 1203-residue protein composed of repetitive motifs and domains. Northern analysis indicates that CSA1 is preferentially expressed during the mycelial growth phase, although a low level of CSA1 mRNA can be detected in the yeast form. As evidenced by indirect immunofluorescence microscopy with mAb 4E1, Csa1p is not randomly distributed over the surface of yeast cells, but localizes predominantly in the growing buds. This suggests that the distribution of Csa1p may be restricted to sites of cell surface elongation. Both heterozygous and homozygous C. albicans csa1Delta mutants are viable. Upon induction of mycelial growth, the number and size of hyphal structures derived from the mutants are similar to those observed in the parental wild-type strain. The physiological role of Csa1p has yet to be determined. However, the presence in Csa1p of repeated cysteine-rich hydrophobic domains with significant sequence similarity to motifs found in surface proteins (Ag2 and Pth11) from two distantly related fungal pathogens (Coccidioides immitis and Magnaporthe grisea respectively) suggests a common function in host interaction.