Vacuolar H+-ATPase (V-ATPase) is a fundamentally important enzyme in eukaryotic cells that is responsible for acidification of endocytic compartments. The B subunits of V-ATPases from mammals and tobacco hornworm have been shown to bind actin filaments. Actin-binding activity by the B subunit is required for targeting V-ATPases to the plasma membrane of osteoclasts. Bacterially expressed B subunit from the yeast Saccharomyces cerevisiae bound actin filaments with a Kd of 195 nmol l(-1). The actin-binding domain of the B subunit was altered by mutations that reduced or eliminated the actin-binding activity. Mutants assembled properly with endogenous yeast subunits when expressed in B subunit-null yeast and bafilomycin-sensitive ATPase activity was not significantly different from yeast transformed with wild-type B subunit. Yeast containing the mutant subunits grew as well at pH 7.5 as wild-type. Screening null yeast or null yeast transformed with wild-type or mutant B subunits with sub-lethal doses of various drugs revealed that yeast containing the mutant B subunits were more sensitive to cycloheximide and wortmannin than those transformed with wild-type B subunits. These results suggest that actin-binding activity confers on the B subunit of yeast a function that is distinct from its role in the enzymatic activity of the proton pump.