Candida albicans ALS3 encodes a large cell-surface glycoprotein that has adhesive properties. Immunostaining of cultured C. albicans germ tubes showed that Als3p is distributed diffusely across the germ tube surface. Two-photon laser scanning microscopy of model catheter biofilms grown using a PALS3-green fluorescent protein (GFP) reporter strain showed GFP production in hyphae throughout the biofilm structure while biofilms grown using a PTPI1-GFP reporter strain showed GFP in both hyphae and yeast-form cells. Model catheter biofilms formed by an als3 Delta/als3 Delta strain were weakened structurally and had approximately half the biomass of a wild-type biofilm. Reintegration of a wild-type ALS3 allele restored biofilm mass and wild-type biofilm structure. Production of an Als3p-Ag alpha 1p fusion protein under control of the ALS3 promoter in the als3 Delta/als3 Delta strain restored some of the wild-type biofilm structural features, but not the wild-type biofilm mass. Despite its inability to restore wild-type biofilm mass, the Als3p-Ag alpha 1p fusion protein mediated adhesion of the als3 Delta/als3 Delta C. albicans strain to human buccal epithelial cells (BECs). The adhesive role of the Als3p N-terminal domain was further demonstrated by blocking adhesion of C. albicans to BECs with immunoglobulin reactive against the Als3p N-terminal sequences. Together, these data suggest that portions of Als3p that are important for biofilm formation may be different from those that are important in BEC adhesion, and that Als3p may have multiple functions in biofilm formation. Overexpression of ALS3 in an efg1 Delta/efg1 Delta strain that was deficient for filamentous growth and biofilm formation resulted in growth of elongated C. albicans cells, even under culture conditions that do not favour filamentation. In the catheter biofilm model, the ALS3 overexpression strain formed biofilm with a mass similar to that of a wild-type control. However, C. albicans cells in the biofilm had yeast-like morphology. This result uncouples the effect of cellular morphology from biofilm formation and underscores the importance of Als3p in biofilm development on silicone elastomer surfaces.