The positive endocochlear potential (EP+) and high K+ concentration of the endolymph in the scala media of the mammalian cochlea are unusual. They have long been assumed to be due to a putative K-pump in the luminal membrane of the marginal cells of the stria vascularis, which were believed to have a negative internal potential. We show that the cell potential is more positive than the EP+, and that the ion pump is conventional Na,K-ATPase, probably in the basolateral membrane. The latter was determined from experiments in which the ionic environment of the strial cells was controlled by perfusion of the perilymphatic space of the cochlea, in the absence of vascular circulation. While the usual EP+ was maintained by normal perfusate, replacement of Na+ by choline resulted in a negative EP, showing that Na,K-ATPase is necessary for the production of EP+. Elimination of K+ as well as Na+ from the perfusate did not change the value of the negative EP, showing that no K-ATPase is involved.