Plasma membrane Ca(2+) ATPases (PMCAs) maintain intracellular Ca(2+) homeostasis and participate in the local regulation of Ca(2+) signaling. Spatially separate demands for Ca(2+) regulation require proper membrane targeting of PMCAs, but the mechanism of PMCA targeting is unknown. Using the PMCA2b carboxyl-terminal tail as yeast two-hybrid bait, we isolated a novel PDZ domain-containing protein from a human brain cDNA library. This protein, named PISP for PMCA-interacting single-PDZ protein, consists of 140 amino acids and contains little else besides a single PDZ domain. Pulldown experiments showed that PISP interacts with all PMCA b-splice forms. PISP was found to be ubiquitously expressed and, in MDCK cells, was present in a punctate pattern throughout the cytosol and at the basolateral membrane. When added to microsomal membranes expressing PMCA4b, PISP was unable to stimulate the PMCA-dependent ATPase activity. Our data suggest that PISP is a transiently interacting partner of the PMCA b-splice forms that may play a role in their sorting to or from the plasma membrane.