Vtc proteins have genetic and physical relations with the vacuolar H(+)-ATPase (V-ATPase), influence vacuolar H(+) uptake and, like the V-ATPase V(0) sectors, are important factors in vacuolar membrane fusion. Vacuoles from vtc1delta and vtc4delta mutants had slightly reduced H(+)-uptake activity. These defects could be separated from Vtc function in vacuole fusion, demonstrating that Vtc proteins have a direct role in membrane fusion. We analyzed their involvement in other membrane trafficking steps and in VATPase dynamics. Deletion of VTC genes did not impede endocytic trafficking to the vacuole. However, ER to Golgi trafficking and further transport to the vacuole was delayed in deltavtc3 cells. In accordance with that, deltavtc3 cells showed a reduced growth rate. Vtc mutations did not interfere with regulated assembly and disassembly of the V-ATPase, but they affected the number of peripheral V(1) subunits associated with the vacuoles. deltavtc3 vacuoles carried significantly more V(1) subunits, whereas deltavtc1, deltavtc2 and deltavtc4 had significantly less. The proteolytic sensitivity of the V(0) subunit Vph1p was different in deltavtc and wild-type cells in vivo, corroborating the physical interaction of Vtc proteins with the V-ATPase observed in vitro. We suggest that Vtc proteins affect the conformation of V(0). They might thereby influence the stability of the VATPase holoenzyme and support the function of its V(0) sector in vacuolar membrane fusion.