Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained approximately 21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively "high mannose-type" glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells.