The macrolide antibiotic bafilomycin A1 is a highly potent and selective inhibitor of all the vacuolar ATPases (V-ATPases). With the aim of obtaining novel analogues specific for the osteoclast subclass of vacuolar ATPase, 31 derivatives of bafilomycin A1 were synthesized and tested for their ability to inhibit differentially the V-ATPase-driven proton transport in membrane vesicles derived from chicken osteoclasts (cOc) and bovine chromaffin granules (bCG). Although none of the new analogues were more potent than the parent compound, the obtained data provided a significant amount of information about the structural requirements for the inhibitory activity of bafilomycin A1. The different effects of a few analogues on the two enzymes could also suggest the possibility of a selective modulation of the V-ATPases in different tissues.