The use of replication-defective adenoviruses to deliver transgenes into hepatocytes seems to be a promising approach to human liver gene therapy. However, the effects that the adenovirus-mediated expression of a foreign gene could have on the expression of other hepatic characteristic genes have not yet been properly examined. We have investigated this problem by using human hepatocytes infected with a recombinant E-1 defective adenovirus that carried a modified lacZ gene. The analysis of the biochemical functionality of transduced cells showed that the use of adenovirus: (1) was a very efficient way to introduce a foreign gene into human hepatocytes (80% transduced cells after 1 h contact, at an MOI of 15; approximately 100% transduced cells at an MOI of 20); (2) allowed the expression of the transgene to levels that enabled cells effectively to use lactose as an energy source; (3) does not affect urea synthesis, plasma protein synthesis and xenobiotic biotransformation activities (1A2, 2A1, 2B6, 3A3/5). Glycolysis was moderately increased (approximately 20%), while gluconeogenesis decreased (approximately 20%) in transduced hepatocyte; moreover, (4) the expression of inducible genes (acute-phase plasma proteins, CYPs) was not impaired in transduced human hepatocytes upon stimulation with IL-6 or methylcholantrene. The results of this research support the idea that efficient expression of transgenes can be achieved in human hepatocytes by means of adenoviral transduction, without altering these characteristic hepatic biochemical functions.