Immunogold labelling of creatine kinase B (BB-CK) and gastric H+/K+-ATPase in the parietal cells of the stomach revealed co-localization of these two enzymes on the apical membrane and the membranes of the tubulovesicular system. Upon fractionation of hog parietal cells, a specific fraction of the BB-CK proteins remained associated with the purified vesicles, in which gastric H+/K+-ATPase is highly enriched. The BB-CK present in this highly purified preparation was able to support pronounced H+/K+-ATPase activity in K+-loaded vesicles in the presence of phosphocreatine and ADP, although only low levels of ATP were measured. In contrast, when pyruvate kinase, phosphoenolpyruvate and ADP were used as an ATP-generating system to sustain similar levels of H+/K+-ATPase activity, ATP levels were more than 10-fold higher. Changing the experimental conditions such that ATP levels were the same for both systems resulted in significantly elevated H+/K+-ATPase activities in the BB-CK/phosphocreatine system in comparison with the pyruvate kinase/phosphoenolpyruvate system. These results indicate that gastric H+/K+-ATPase has preferential access to ATP generated by creatine kinase co-localized on the membranes of the vesicles.