The model plant tobacco (Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.