The structurally related ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases fulfill overlapping yet non-redundant functions as key regulators of cellular DNA damage responses. We recently showed that ATM phosphorylates the cyclic AMP response element-binding protein, CREB, following exposure to ionizing radiation (IR) and other DNA-damaging stimuli. Here, we show that a phospho-specific antibody recognizing the major ATM phosphorylation site in CREB cross-reacts with SV40 large tumor antigen (LTag), a multifunctional oncoprotein required for replication of the SV40 minichromosome. The relevant IR-induced phosphorylation site in LTag recognized by phospho-CREB antibody was mapped to Ser-120. IR strongly induced the phosphorylation of Ser-120 in an ATM-dependent manner in mouse embryo fibroblasts. Infection of African green monkey CV1 cells with SV40 resulted in the activation of ATM and phosphorylation of LTag and endogenous ATM substrates. Infection-induced LTag phosphorylation correlated with the onset of DNA replication, was ATM-dependent, and peaked when viral DNA levels reached their maximum. SV40 replication in CV1 cells required an intact LTag Ser-120 phosphorylation site and was inhibited following transfection with ATM small interfering RNA suggesting that ATM is required for optimal SV40 replication in primate cells. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.