Retroviral vectors have been widely studied as vehicles for hepatocyte gene therapy, but they are limited by an inability to infect nondividing cells and the need for prolonged cell culture. Two replication deficient herpes simplex viral vectors (HSV) were constructed with the marker genes Zac-Z//%galactosidase (HSVlac) or human-growth hormone (HSVhGH) to determine the efficiency of HSV gene transfer into adult human hepatocytes. Hepatocytes were isolated by collagenase perfusions and density centrifugation from liver wedge biopsy specimens obtained from six patients. After exposure to HSV (0, 50,000, and 500,000 viral particles/ 10' hepatocytes) for 20 minutes, 1 hour, or 2 hours, the hepatocytes were washed and placed in culture. Hepatocytes transduced with HSVlac were fixed at 24 hours and histochemically stained with X-gal, and media from HSVhGH-transduced cells were assayed at 48 hours by radioimmunoassay for hGH. After a 20-min-Ute exposure at a multiplicity of infection of 0.5 (1 viral particle per 2 hepatocytes), greater than 35% of the hepatocytes expressed the lac-2 gene (>TO% efficiency). hGH was also detected in the media from HSVhGH-transduced cells, showing that proteins coded for by foreign genes are not only expressed by transduced cells but are also secreted. Isolated liver perfusions using HSVlac were also performed in Fischer rats. A 20-minute isolated perfusion using 5 x 10" viral particles resulted in expression of the P-galactosidase gene in the rodent livers 72 hours later without histological signs of tissue injury. HSV vectors are potentially powerful tools for gene therapy of human liver disease, because they are efficient and rapid vehicles for gene transfer. Ex uiuo modified hepatocytes theoretically may be ready for reinfusion within 100 Abbreviations: HSV, herpes simplex viral vectors; HSVlac, lac-Z/fi-galactosidase; HSVhGH, human growth hormone; EGTA, ethyleneglycodiaminetetraacetate; FCS. fetal calf serum; MOI, multiplicity of infection.