Liver-directed gene transfer offers new perspectives for the treatment of inherited metabolic diseases and disorders of lipoprotein metabolism. Potent expression cassettes for transgenes in the liver may optimize gene transfer efficiency and improve the therapeutic index of gene transfer vectors. An E(1)-deleted adenovirus comprising the hepatocyte specific 256-base pair (bp) human apolipoprotein A-I (apo A-I) promoter, the genomic human apo A-I DNA, and four human apo E enhancers (AdA-I.gA-I.4xapoE) was associated with low hepatotoxicity, high transgene DNA persistence and absence of promoter shut-off, resulting in human apo A-I plasma levels above 100 mg/dl for 35 days in C57BL/6 mice. In the present investigation, the human apo A-I promoter was compared to the murine small nuclear RNA U1b, the human apolipoprotein C-II (apo C-II), and the human alpha(1) antitrypsin (hAAT) promoters and the effect of copy number and position of liver-specific human apo E enhancers in 16 adenoviral constructs was evaluated. The vector containing the 1.5-kb hAAT instead of the apo A-I promoter (AdhAAT.gA-I.4xapoE) induced 3.7-fold (p < 0.01) more human apo A-I reaching plasma levels above 300 mg/dl for 35 days. The composition of the expression cassette was a major determinant of human apo A-I transgene DNA copy number at day 35. Hepatotoxicity after adenoviral gene transfer was dependent on the promoter and the number of enhancers, and was higher with the enhancers in a 5' position. The combination of the hAAT promoter and four copies of the human apo E enhancer appears to be the expression cassette of choice for hepatocyte-specific overexpression of transgenes after gene transfer.