TNF is a multifunctional protein that is secreted by activated macrophages and is believed to have antiviral properties. We reported previously that certain murine fibroblasts infected with group C human adenoviruses become sensitive to cytolysis by TNF, and that a 14,700 m.w. (14.7K) protein encoded by the E3 transcription unit protects the cells from TNF cytolysis. We now report the mapping of the adenovirus genes that induce sensitivity to TNF cytolysis. Experiments using hydroxyurea or 1-beta-D-arabinofuranosylcytosine showed that sensitivity is conferred by an early gene. Further mapping was done by infecting cells with double mutants that lack both the TNF protection function of the E3-14.7K protein and various other early genes and testing infected cells for TNF sensitivity. Using this method we found that none of the genes in regions E1B, E3, E4, or VA1-RNA are involved in creating the TNF-sensitive cellular phenotype. However, TNF did not lyse cells infected with dl312, an adenovirus mutant that lacks the 14.7K gene plus region E1A, suggesting that the E1A proteins are required to induce sensitivity to TNF. Mutants dl237 and 13SWT, which produce only the 12S (243R) or 13S (289R) E1A proteins, respectively, do render infected cells sensitive to TNF, indicating that both proteins provide this function. Finally, E1A-transfected cells, which are sensitive to TNF killing, do not become more sensitive when infected with dl312, thus indicating that E1A alone among adenovirus genes is responsible for the TNF susceptibility of infected cells.