In the stria vascularis (SV), it is known that the Na(+)-K(+)-ATPase is expressed abundantly and its activity in the basolateral membrane of marginal cells is high. Ouabain, an inhibitor of the Na(+)-K(+)-ATPase, causes not only a decline in the endocochlear DC potential but also acute vacuolar formation in marginal cells. We studied the ionic mechanisms underlying the ouabain-induced vacuolar formation in marginal cells using perilymphatic perfusion in guinea pigs. Perilymphatic perfusion with 1 mM ouabain dissolved in the artificial perilymph for 50 min caused many vacuoles of a wide range of sizes in the apical cytoplasm of marginal cells, the bulging of marginal cells into the scala media and strial volume increase. Removal of K(+) from the perilymph reduced the proportion of vacuoles and strial thickening, but the bulging of marginal cells remained. In contrast, the sizes of vacuoles were drastically reduced and extrusion of marginal cells into the scala media could not be observed in the absence of perilymphatic Na(+). Furthermore, the total volume of SV was obviously reduced in comparison with the control. These results indicate that perilymphatic Na(+) and K(+) are responsible for these morphological changes caused by ouabain, and that perilymphatic Na(+) plays an important role in the cellular volume regulation in SV in the presence of ouabain. It is supposed that the transport system of perilymphatic Na(+) and K(+) into marginal cells may contribute to vacuolar formation when ouabain is administered. Regarding Na(+), we hypothesize two possibilities for the perilymphatic Na(+) transporting pathway as follows. Na(+) in the perilymph could enter the endolymph via Reissner's membrane or the basilar membrane; Na(+) in the endolymph would then be taken up by marginal cells via the apical membrane and secreted into the intrastrial space by Na(+)-K(+)-ATPase in the basolateral membrane. Another, less likely, possibility is that Na(+) in the perilymph is transported into basal cells or fibrocytes in the spiral ligament, then into intermediate cells via gap junctions, and finally secreted into the intrastrial space via Na(+)-K(+)-ATPase of intermediate cells. Regarding K(+), it is expected that the K(+) recycling pathway plays a role in ouabain-induced vacuolar formation in marginal cells.