Many hepatocyte-specific genes are expressed heterogeneously in the liver lobule depending on the location of the hepatocytes in relation to the inflow or outflow of portal blood (i.e., periportal or perivenous). For example, albumin is expressed in all hepatocytes but more so in the periportal zone, cytochrome P-450IIE1 is exclusively expressed in the perivenous zone and glutamine synthetase is limited to one or two cell layers next to the terminal hepatic venule. Additionally, hepatic damage caused by several xenobiotics, including carbon tetrachloride, is more severe in the perivenous zone. We have isolated highly enriched perivenous and periportal hepatocytes by means of a digitonin-collagenase perfusion method and transplanted them separately into the spleens of syngeneic rats. After transplantation, hepatocyte-specific gene expression in the transplanted perivenous and periportal cells was monitored for up to 13 mo with in situ hybridization to detect the specific gene transcripts (mRNAs). We also studied the effects of carbon tetrachloride administration on transplanted periportal cells by comparing them with intrasplenic transplanted periportal hepatocytes without carbon tetrachloride treatment. Our results showed that: (a) both transplanted perivenous and periportal hepatocytes could survive and proliferate in the splenic microenvironment for a prolonged period; (b) long-term-transplanted periportal hepatocytes in spleen could eventually express a high level of cytochrome P-450IIE1 mRNA in all transplanted hepatocytes and could express glutamine synthetase mRNA in only about 5% to 10% of them, specifically those hepatocytes located adjacent to splenic blood vessels. It is noteworthy that periportal hepatocytes in situ normally do not express the glutamine synthetase gene and express only a low level of cytochrome P-450IIE1 mRNA; and (c) carbon tetrachloride yielded different toxic effects on transplanted periportal hepatocytes at day 3 and mo 8. Necrosis was seen only when transplanted periportal hepatocytes expressed a high level of cytochrome P-450IIE1 mRNA by mo 8.