The vacuolar H+-ATPase is inhibited with high specificity and potency by bafilomycin and concanamycin, macrolide antibiotics with similar structures. We previously reported that mutation at three residues in subunit c of the vacuolar ATPase from Neurospora crassa conferred strong resistance to bafilomycin but little or no resistance to concanamycin (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). We have identified additional mutated sites in subunit c that confer resistance to bafilomycin. Furthermore, by subjecting a resistant mutant to a second round of mutation we isolated strains with increased resistance to both bafilomycin and concanamycin. In all of these strains the second mutation is also in subunit c, suggesting it forms at least part of the concanamycin binding site. Site-directed mutagenesis of the gene encoding subunit c in Saccharomyces cerevisiae showed that single mutations in each of the residues identified in one of the double mutants of N. crassa conferred resistance to both bafilomycin and concanamycin. Mutations at the corresponding sites in the VMA11 and VMA16 genes of S. cerevisiae, which encode the c' and c" subunits, did not confer resistance to the drugs. In all, nine residues of subunit c have been implicated in drug binding. The positions of these residues support a model in which the drug binding site is a pocket formed by helices 1, 2, and 4. We hypothesize that the drugs inhibit by preventing the rotation of the c subunits.