We (41) 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 30 h postexposure (PE) to the virus. Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semiquantitative 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 approximately 48 h, and <20% of the total expressed NKCC protein are in the plasma membrane. The remainder (approximately 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 (approximately 75%) within 24 h 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; and 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72 h 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.