Extracellular glutathione peroxidase (eGPx) is a secreted selenoenzyme with glutathione peroxidase activity. eGPx protein and activity are found in blood plasma and other extracellular fluids. eGPx in plasma is predominantly derived from the proximal tubules of kidneys in humans. Two types of human proximal tubule cells were cultured on semi-permeable polycarbonate membranes to determine whether these cells secrete eGPx in a polarized direction. The immortalized human proximal tubule HK-2 cells and primary human proximal tubule cells formed confluent monolayers when cultured on these membrane inserts in culture dishes, as evidenced by transepithelial resistance. Both cell lines also constituted a barrier to diffusion of a fluoresceinated dextran of 75 kDa, a size similar to eGPx homotetramers. Six- to twelve-fold more ³⁵S-Met-labeled eGPx was immunoprecipitated from the basolateral media than from the apical media of both cell lines, indicating basolateral secretion of eGPx. eGPx was immunolocalized to the extracellular fluid at the basolateral surface of proximal tubules in human kidney. These data support the conclusion that eGPx is secreted through the basolateral membrane of human kidney proximal tubule cells into the extracellular fluid of the kidney, and from there enters blood plasma.