A synthetic GAL4-responsive promoter consisting of five GAL4-binding sites and a TATA box (GAL4/TATA) was evaluated for its transcriptional activity in an adenoviral backbone using luciferase as the reporter. Basal luciferase activities in vitro were the same for cells infected with either adenovirus-containing luciferase cDNA driven by GAL4/TATA (Ad/GT-Luc) or adenovirus-containing luciferase cDNA not driven by a promoter (Ad/PO-Luc). In vitro induction of GAL4/TATA by coinfection of cells with adenovirus expressing the GAL4/VP16 fusion protein (Ad/3-phosphoglycerate kinase (PGK)-GV16) was dose-dependent and reached as high as 4 x 10(4)- to 9 x 10(4)-fold above basal levels when GAL4/TATA and GAL4/VP16 were delivered at a ratio of 10:1. In vivo studies in Balb/c mice showed no detectable luciferase activities in liver or other tissues examined in mice infused with either Ad/GT-Luc or Ad/PO-Luc. High levels of luciferase activity were, however, elicited when animals were infused with Ad/GT-Luc and Ad/PGK-GV16. Together, these results suggest that combination of the GAL4 gene regulatory system with adenovirally mediated in vivo gene delivery may be applicable to the in vivo evaluation of promoter activities and in vivo targeting of gene expression.