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Summary.:  Hepatocyte preparations highly enriched in cells from either the periportal or the perivenous zone of the liver acinus were prepared using a digitonin/collagenase perfusion method. Five enzymes of cysteine metabolism were assayed in both periportal and perivenous preparations. The ratios of periportal to perivenous activity were 0.76, 0.60, 0.81, 1.62, and 1.01 for cysteine dioxygenase, cysteinesulfinate decarboxylase, γ-glutamylcysteine synthetase, cystathionase, and asparate (cysteinesulfinate) aminotransferase, respectively. Only cysteinesulfinate decarboxylase activity was significantly different between periportal and perivenous cells. In incubations with 2 mmol/L [35S]cysteine, total cysteine catabolism ([35S]taurine plus [35S]sulfate) between periportal and perivenous cells was not different, which is consistent with the observation of similar cysteine dioxygenase activity across the hepatic acinus. Consistent with the lower cysteinesulfinate decarboxylase activity in periportal cells, 16% of the total catabolism of [35S]cysteine in periportal cells resulted in taurine synthesis compared to 28% in perivenous cells. A lower rate of [35S]glutathione synthesis was observed in periportal cells compared to perivenous cells, but γ-glutamylcysteine synthetase activity was not significantly different between perivenous and periportal cells. Cysteinesulfnate decarboxylase can be added to the list of enzymes whose activities are markedly enriched in perivenous cells.