In preparation for studies using gene transfer, we have identified transcriptional control elements which are active in primary rat hepatocytes. We used plasmids which were constructed so that the promoter or enhancer of interest initiated transcription of the bacterial chloramphenicol acetyltransferase (CAT) gene. Plasmids were introduced into primary rat hepatocytes in culture, into Hep G2 cells and other human and animal cell lines and into bone marrow stromal cells, and CAT activity was assayed after 48 hr. In primary rat hepatocytes, the highest CAT activity was obtained with plasmids carrying the Rous sarcoma virus long terminal repeat (pRSVCAT), or the SV40 early region promoter and enhancer (pSV2CAT). Hepatocytes carrying the murine cytomegalovirus immediate early promoter (pUCRNmCMVX/HCAT) also had appreciable CAT activity. No CAT activity was detected in rat hepatocytes carrying pSVOCAT (a promoterless construct), pUCRNtKCAT (herpes simplex thymidine kinase gene promoter), pLPVCAT (lymphocytotrophic papovavirus promoter) and pHBV1CAT (hepatitis B virus enhancer and core gene promoter). Therefore, for future studies of gene transfer in primary rat hepatocytes, the Rous sarcoma virus long terminal repeat or the SV40 early region promoter and enhancer can be effectively used to drive gene expression. Hep G2 cells carrying pHBV1CAT had high CAT activity. Hep G2 cells carrying pHBV2CAT (similar to pHBV1CAT, but with the hepatitis B virus sequences in reverse orientation with respect to the CAT sequences) and pHBV3CAT (similar to pHBV2CAT, but hepatitis B virus sequences are separated from the CAT sequences by about 700 bases) also expressed CAT activity, but not as strongly as with pHBV1CAT.(ABSTRACT TRUNCATED AT 250 WORDS)