Vectorial Na⁺ reabsorption across the proximal tubule is mediated by apical entry of Na⁺, primarily via Na⁺/H⁺ exchanger isoform 3 (NHE3), and basolateral extrusion via the Na⁺ pump (Na⁺-K⁺-ATPase). We hypothesized that regulation of Na⁺ reabsorption should involve not only the activity of the basolateral Na⁺-K⁺-ATPase but also the apical NHE3, in a concerted manner. In order to generate a cell line that overexpresses Na⁺-K⁺-ATPase, OK cells were transfected with the rodent Na⁺-K⁺-ATPase ₁-subunit (pCMV ouabain vector), and native cells used as a control. The existence of distinct functional classes of Na⁺-K⁺-ATPase in wild type and transfected cells was confirmed by the inhibition profile of Na⁺-K⁺-ATPase activity by ouabain. In contrast to wild type cells, transfected cells exhibited two IC_50s for ouabain. The first was similar to the IC₅₀ of control cells; the second IC₅₀ was 2 logs greater than the first, consistent with the presence of both rat and opossum 1 isozymes. It is shown that transfection of OK cells with Na⁺-K⁺-ATPase increased both Na⁺-K⁺-ATPase and NHE3 activities. This was associated with overexpression of the Na⁺-K⁺-ATPase ₁-subunit and NHE3 in transfected OK cells. The abundance of the Na⁺-K⁺-ATPase ₁-subunit was slightly lower in transfected OK cells. In conclusion, the increase in both expression and function of Na⁺-K⁺-ATPase found in cells transfected with the rodent Na⁺ pump ₁ cDNA is expected to stimulate apical Na⁺ influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.