Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3Δ/als3Δ mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related Candida dubliniensis. The 3D9 epitope was detected only in C. albicans, demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis. Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.