ABSTRACT—In vitro studies were carried out on isolated rat hepatocytes to examine further the proposed cytoprotective actions of prostaglandins (PG) using carbon tetrachloride (CCl4) as the toxic agent. Isolated hepatocytes, prepared by collagenase, were cultured in Leibowitz-15 medium. Following preincubation, CCl4(300 or 150 μg/ml) was added to the hepatocytes. Treatment with Indomethacin (INDO), 16, 16-dimethyl-PGE2(PGE2) and prostacyclin (PGI2) was assayed in the cultures. Cell damage was measured by lactic dehydrogenase (LDH) release. 6-keto-PGF1α was measured in the supernatant by direct radioimmunoassay. The results showed PGI2 (30.0 ng/ml) treatment 30 min after CCl4(300 μg/ml) addition to be highly protective (p<0.001 versus CCl4 control). PGE2(3 ng/ml) showed similar protection (p<0.001). INDO (2 μg/ml) following CCl4(150 μg/ml) demonstrated increased cell death (p<0.001). INDO (0.5 μg/ml) reduced 6-keto-PGF1α production (p<0.05). Low dose ethanol (1.5 μg/ ml) increased 6-keto-PGF1α production (p<0.05). Ethanol (1.5 μg/ml), added to stimulate endogenous PG production, was cytoprotective when added prior to CCl4 (p<0.01). This protection was suppressed by INDO. Ethanol added after CCl4 was not protective. We conclude that exogenously added PGI2and PGE2 are cytoprotective in this in vitro model and that endogenous PG production may play a protective role in the initial stages of cellular damage.