Infection of quiescent cells with the DNA tumor virus simian virus 40 induces expression of the cellular thymidine kinase (TK) gene a minimum of 10- to 20-fold, and this induction depends upon the viral protein large T antigen (T-Ag). To define both human TK promoter elements and T-Ag functional domains required for transcriptional induction, we have established a system in which stable Rat-1 transfectants harboring TK promoter-luciferase hybrid genes are infected with recombinant adenoviruses expressing either wild-type or mutant forms of T-Ag and luciferase expression is measured as an indicator of promoter activity. The results show that (i) a 135-bp TK promoter fragment is activated 10- to 15-fold by viral infection; (ii) this activation is the result of both T-Ag-dependent and -independent mechanisms; (iii) the T-Ag pRb family-binding domain, but not the p53-binding, helicase, or ATPase domain, is required for activation; and (iv) activation is severely diminished with a TK promoter fragment in which E2F-like-binding sites have been removed. These data demonstrate a requirement for both an E2F-related factor and a pRb family member in activation of the TK promoter by T-Ag. This contrasts with the promiscuous activation of many cellular and viral genes by T-Ag, which is independent of its ability to bind pRb.