Primary hepatocytes are an important in vitro model for studying metabolism in man. Caspase-9 and Bcl-2-associated X protein (Bax) are regulators of the apoptotic pathway. Here we report on the translocation of procaspase-9 and Bax from cytoplasm to nuclei as well as on dispersion of mitochondria; these processes occur after isolation of primary hepatocytes. The observed changes appear similar to those at the beginning of apoptosis; however, the isolated hepatocytes are not apoptotic for the following reasons: (1) cells have a normal morphology and function; (2) the mitochondria are energized; (3) there is no apoptosis unless it is induced by, e.g., staurosporine or nodularin. Staurosporine does not trigger apoptosis through activation of caspase-9, as its activity is detected later than that of caspase-3. We propose that the translocation of procaspase-9 and Bax into the nuclei reduces the ability to trigger apoptosis through the intrinsic apoptotic pathway. The shifts of procaspase-9 and Bax are reversible in the absence of the apoptotic trigger; the spontaneous reversion was confirmed experimentally for procaspase-9, whereas Bax shifted from the nuclei to the cytosol and mitochondria after the initiation of apoptosis. To distinguish this process from apoptosis, we call it preapoptotic cell stress response. It shares some features with apoptosis; however, it is reversible and apoptosis has to be induced in addition to this process. Conclusion: Knowledge on preapoptotic cell stress response is important for assessing the quality of the cells used in cell therapies, in regenerative medicine, and of those used for modeling metabolic processes. (HEPATOLOGY 2010;51:2140-2151 C ell cultures, especially those of primary cells, are important models for studying biochemical and physiological phenomena; they are also used in cell therapies and in regenerative medicine. Ideally, the metabolism of isolated cells should not differ from the metabolism within the cells of intact tissues; therefore, the primary cell cultures are thought to be the closest models of in vivo processes. The primary cell cultures of isolated hepatocytes are being used as models to study drug metabolism or drug effects on the liver.1 Nevertheless, some metabolic changes occur upon culturing primary hepatocytes. Expression of cytochromes P450 (CYP) is especially well studied, because of their involvement in drug metabolism. The CYP messenger RNA (mRNA) levels of isolated hepatocytes are similar to those of liver 2 ; however, they decline progressively during the first days in culture.3 Also, there are significant perturbations of genes encoding for antioxidant enzymes, heat shock proteins, nitric oxide synthase, and methionine adenosyltransferase following the isolation and culture of hepatocytes. 2As a consequence, the use of primary cultured hepatocytes in drug metabolism studies is confined to the first days in culture. 4 Although the metabolic state of isolated hepatocytes is extensively studied, there are no data on modulation of apoptotic ma...