A series of simian virus 40-immortalized hepatocyte cell lines which are heterogeneous with regard to expression of albumin protein and RNA were characterized for their ability to transcribe the albumin gene. Nascent chain extension assay showed that albumin RNA levels in these cells were determined predominantly at the transcription level. The albumin promoter and enhancer sequences were fused to the bacterial chloramphenicol acetyltransferase gene; the ability of the resulting expression constructs to drive chloramphenicol acetyltransferase expression after transfection into these hepatocyte cell lines was measured. The activity of the albumin promoter and enhancer constructs in primary hepatocytes was also measured. The albumin promoter was expressed differentially in these cells; however, no correlation was found between the transcriptional efficiency of the transfected albumin promoter and endogenous albumin transcription. The albumin enhancer was functional in some but not all albumin-positive cells. The minimal albumin enhancer was mapped to a 330-base pair fragment extending from -9.94 kilobases (kb) to -10.27 kb; three elements within this fragment recently shown to be necessary for enhancer function in a murine hepatocyte cell line were also essential for albumin enhancer function in the rat hepatocyte cell line CWSV1. A transcriptional silencer was identified which could suppress the expression of the homologous albumin promoter and the heterologous herpes simplex virus thymidine kinase promoter. Preliminary analysis localized the albumin silencer between -11 and -12 kb. Our results suggest that multiple regulatory sequences may act cooperatively to determine efficient tissue-specific expression of the albumin gene.