We previously reported that Na,K,Cl cotransporter (NKCC) function and microsomal protein expression are both dramatically reduced late in human cytomegalovirus (HCMV) infection of a human fibroblast cell line (MRC-5). We now report DNA microarray data showing that no significant HCMV dependent NKCC gene repression can be detected 30h post-exposure to the virus (30h PE). Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semi-quantitative immunoblotting, and confocal microscopy, to investigate the possibility that intracellular redistribution of the NKCC protein after HCMV infection could be a cause of the HCMV-induced loss of NKCC ion transport function. Our results show that the lifetime of plasmalemmal NKCC protein in quiescent, uninfected MRC-5 cells is ~48h and less than 20% of the total expressed NKCC protein is in the plasma membrane; the remainder (~80%) was detected as diffusely distributed, small punctate structures in the cytoplasm. Following HCMV infection: 1) NKCC protein expression in the plasmalemma was sharply reduced (~ 75%) within 24h PE and thereafter continued to slowly decrease; 2) total cellular NKCC protein content remained unchanged or slightly increased during the course of the viral infection; 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72h PE). Thus, our results imply that in the process of productive HCMV infection, NKCC protein continues to be synthesized, but instead of being delivered to the plasma membrane, it is clustered in a large, detergent soluble perinuclear structure.