In several in-vivo settings, prolonged alterations in the rate of apical Na⁺ entry into epithelial cells alters the ability of these cells to reabsorb Na⁺. We have previously modeled this load dependence of transport in A6 cells by either decreasing Na⁺ entry via apical Na⁺ removal or amiloride, or enhancing Na⁺ entry by chronic short-circuiting (Am J Physiol 270:C600-C607, 1996). Inhibition of Na⁺ entry by either method was associated with striking down-regulation of transport rate as measured by short-circuit current (I_sc), which recovered to basal levels of transport over a period of hours. Conversely, up-regulation of Na⁺ entry by short-circuiting, resulted in a sustained increase in transport rate which also returned to basal levels over a period of hours. The current studies were undertaken to determine whether these conditions were associated with alterations in either the whole cell content or apical membrane distribution of sodium channel (ENaC) subunits or on basolateral expression of either of the subunits of the Na⁺/K⁺ATPase. We compared these effects to those achieved by chronic up-regulation of Na⁺ transport by aldosterone. Whole cell levels of ENaC subunits were measured by immunoblot following 18h inhibition of Na⁺ entry achieved by either TMA replacement of Na⁺ or apical amiloride, or after 18h increase in Na⁺ entry achieved by chronic short-circuiting. None of these maneuvers significantly altered the whole cell content of any of the ENaC subunits compared to control cells. We then examined the effects of these maneuvers on apical membrane ENaC expression using domain-specific biotinylation and immunoblot. Inhibition of Na⁺ entry by either method was associated with a profound decrease in apical membrane ENaC without significant changes in apical membrane or ENaC amounts. Restoration of apical Na⁺ and/or removal of amiloride resulted in return of I_sc to control levels over 2 hours and coincided with return of apical ENaC to control levels without change in apical or ENaC. Stimulation of Na⁺ transport by short-circuiting, in contrast, did not significantly alter apical membrane composition of any of the ENaC subunits. Basolateral expression of Na⁺/K⁺-ATPase was also measured by biotinylation and immunoblot and was unchanged under all conditions. Aldosterone increased basolateral expression of the subunit of Na⁺/K⁺-ATPase. These results suggest that chronic down-regulation of transport is mediated, in part, by a selective decrease in apical membrane ENaC expression, consistent with our previous observations of non-coordinate regulation of ENaC expression under varying transport conditions in A6 cells. The chronic increase in the rate of Na⁺ entry is not associated with any of the changes in transporter density at either apical or basolateral membrane seen with aldosterone suggesting that these two mechanisms of augmenting transport are completely distinct.