Proteinase inhibitor 9 (PI-9, SerpinB9) is the only known human intracellular granzyme B inhibitor. Whether expression of PI-9 is sufficient to block cytolysis induced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells remains controversial. To evaluate the roles of PI-9, we isolated and tested three lines of stably transfected HeLa cells expressing wild-type PI-9 and one line expressing an inactive mutant PI-9. Expressions of wild-type PI-9, but not the inactive mutant PI-9, inhibited cytolysis induced by human NK92 and NKL natural killer cells. Expression of high levels of PI-9 is therefore sufficient to protect human cells against NK cell-mediated cell death. Using two assays, we show that expressing wild-type PI-9, but not the inactive mutant PI-9, blocks Fas/Fas ligand (Fas/FasL)-mediated apoptosis. PI-9 expression has no effect on etoposide-induced apoptosis. HeLa cells exhibiting substantial resistance to Fas/FasL-mediated apoptosis contain 2- to 3-fold higher PI-9 levels than HCT116 human colon cancer cells and 2- to 3-fold lower PI-9 levels than MCF7/ERHA breast cancer cells, in which PI-9 is strongly induced by estrogens, and by tamoxifen. Expression of increasing levels of PI-9 in target cells may progressively inhibit immune surveillance by blocking NK and CTL-induced cytotoxicity through the perforin/granzyme pathway and then through the Fas/FasL pathway.