The molecular basis for pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis remains unknown. WI-1 is a major cell wall protein of B. dermatitidis yeasts and is a recognition target of both humoral and cell-mediated immunity. As an initial study to determine if WI-1 might be linked to virulence of B. dermatitidis, we quantified WI-1 expression on three genetically related strains that differ in their virulence for mice: wild-type virulent ATCC strain 26199, mutant ATCC strain 60915 (which is 10,000-fold reduced in virulence), and mutant ATCC strain 60916 (which is avirulent). Two principal alterations in WI-1 expression were observed in the mutants. First, the mutants express more WI-1 on their surface, as quantified by flow cytometry with monoclonal antibody to WI-1 and by radioimmunoassay, but the WI-1 on their cell wall is less extractable than that on the wild-type strain. Second, the mutants shed less WI-1 during culture and demonstrate impaired processing of shed WI-1. Surface alterations in WI-1 were accompanied by significant differences in the binding of the virulent and mutant strains to human monocyte-derived macrophages. Attachment of yeasts to macrophages paralleled and was proportional to the expression of WI-1. Compared with wild-type yeasts, both mutants bound to macrophages more rapidly and in two- to threefold-greater magnitude. Furthermore, about 75% of yeast binding to macrophages was inhibited by a Fab anti-WI-1 monoclonal antibody. These results suggest that altered WI-1 expression on attenuated and avirulent mutant B. dermatitidis yeasts greatly facilitates macrophage recognition and binding of yeasts and, in turn, may contribute to more rapid ingestion and killing in the host.