Adenovirus E1A proteins can induce quiescent cells to enter S-phase and also affect the expression of cellular genes including various cell cycle regulators. Here we show that human cdc25A, a tyrosine phosphatase involved in regulation of the G1/S-phase transition of the cell cycle, is a target of the adenovirus E1A protein in virus-infected human fibroblasts. Expression of E1A in quiescent fibroblasts leads to a rapid increase in cdc25A phosphatase activity and also increases both cdc25A and cyclin E gene expression. Inhibition of cdc25A function by antibody injection prevents virus-induced entry into S-phase. These results indicate that induction of high levels of cdc25A and its potential positive regulator cyclin E mediates the ability of E1A to induce S-phase in the presence of antiproliferative signals.