During the course of lytic infection, the adenovirus major late promoter (MLP) is induced to high levels after replication of viral DNA has started. We had previously shown that sequence elements located downstream of the MLP start site were implicated in this late-specific transcriptional activation (DE1, between +85 and +98; DE2, between +100 and +120). Two positive transcription factors involved in this activation have been detected. DEF-A, which specifically binds to DE1 and also to the 3' portion of DE2 (DE2a), and DEF-B, which interacts with the 5' part of DE2 (DE2b). When present together, these two proteins cooperatively assemble onto the DE2 element. We now report the purification of DEF-B and show that it is identical to the product of the adenovirus IVa2 gene product. This conclusion is based on microsequence analysis of DEF-B as well as on the inhibitory effect of antibodies against IVa2 on the DNA-binding activity of DEF-B and also on DE-dependent in vitro transcription. In addition, we show that bacterially synthesized IVa2 protein binds to the DE sequences with the same specificity as DEF-B. Finally, in transfected cells, a recombinant IVa2 protein stimulates MLP activity in a DE-dependent fashion. The physiological implications of these findings are discussed.