Adenovirus E1B proteins (19,000-molecular-weight [19K] and 55K proteins) inhibit apoptosis and cooperate with adenovirus E1A to induce full oncogenic transformation of primary cells. The E1B 19K protein has previously been shown to be capable of activating transcription; however, the underlying mechanisms are unclear. Here, we show that adenovirus infection activates the c-Jun N-terminal kinase (JNK) and that the E1B gene products are necessary for adenovirus to activate JNK. In transfection assays, we show that the E1B 19K protein is sufficient to activate JNK and can strongly induce c-Jun-dependent transcription. Mapping studies show that the C-terminal portion of E1B 19K is necessary for induction of c-Jun-mediated transcription. Using dominant-negative mutants of several kinases upstream of JNK, we show that MEKK1 and MKK4, but not Ras, are involved in the induction of JNK activity by adenovirus infection. The same dominant-negative kinase mutants also block the ability of E1B 19K to induce c-Jun-mediated transcription. Taken together, these results suggest that E1B 19K may utilize the MEKK1-MKK4-JNK signaling pathway to activate c-Jun-dependent transcription and demonstrate a novel, kinase-activating activity of E1B 19K that may underlie its ability to regulate transcription.