To evaluate the ability of replication-deficient, recombinant adenovirus vectors to transfer genes to human tumor cells in vivo, adenovirus vectors containing the Escherichia coli lacZ (Ad.RSVβgal) gene (coding for β-galactosidase; used as a cell marker for gene transfer) or the human α1-antitrypsin (Ad-α1AT) cDNA (used as an example of a secreted protein) were administered intraperitoneally to nude mice with human malignant mesothelioma cell (H-MESO-1) malignant ascites. Preliminary in vitro studies showed that both vectors effectively transferred genes to H-MESO-1 cells. Tumor cells recovered from ascites of animals intraperitoneally administered a control adenovirus revealed no evidence of β-galactosidase (β-gal) activity 3 or 14 days later. In contrast, β-gal activity was detected at the same time points in tumor cells from animals receiving intraperitoneal Ad.RSVβgal. Flow cytometric quantification of β-gal activity in recovered cells showed <3% β-gal-positive cells in animals administered control virus, but in animals administered intraperitoneal Ad.RSVβ gal there was a mean of 71 ± 18% positive cells at 3 days and 56 ± 27% at 14 days. Human α1AT was not detected by enzyme-linked immunosorbent assay (ELISA) in ascites of animals receiving a control virus; however, in ascites of animals administered Ad-α1AT, 21,000 ± 3,800 ng/ml of human α1AT was detected at 3 days and 4,900 ± 1,700 ng/ml at 14 days. These data demonstrate that replication-deficient recombinant adenovirus vectors can be used to transfer genes to malignant cells in vivo and suggest a new strategy for genetic modification for antitumor therapy.