Phase partitioning analyses of a brush border membrane preparation obtained with a divalent cation precipitation procedure (Am J Physiol 246:F853-F858, 1984) confirmed that Na+/H+ antiport activity was localized primarily to the brush border membrane of the rabbit proximal tubular epithelial cell. This analysis also indicated that antiport activity was associated with membrane populations that appeared to be derived from cytoplasmic structures. However, since the starting point of the analysis was a partially-purified brush border sample rather than a total membrane sample, it was not possible to discern the magnitude of the potential cytoplasmic pool of antiport activity. We have now used a three dimensional analytical fractionation procedure, based on differential centrifugation, equilibrium density gradient centrifugation, and partitioning in an aqueous polymer 2-phase system, to survey the subcellular distribution of Na/H antiport activity in rat kidney cortex. Roughly 53% of the recovered antiport activity could be assigned to a population of brush border membrane vesicles characterized by a 15-fold enrichment of maltase. An additional 26% of the recovered activity could be assigned to a group of three membrane populations whose biochemical characteristics appeared equally consistent with origins in distinct microdomains of the brush border membrane and with origins in microdomains of the Golgi complex involved in the assembly or recycling of brush border membrane constituents. Therefore, depending on the identities of membranes which contained the secondary pool of Na+/H+ antiport activity, no more than one-third of the total recoverable Na+/H+ antiport activity could be assigned to cytoplasmic membranes of the proximal tubular epithelium.