The ATP-dependent binding of simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication is an essential step in the initiation of SV40 DNA synthesis. Previous studies indicated that the ATP-dependent complex consists of a double hexamer of T antigen at the origin. The binding reaction and the subsequent unwinding of the duplex DNA from the origin were examined using a gel mobility shift assay. T antigen bound to the core origin cooperatively in the presence of ATP. In the presence of human single-stranded DNA-binding protein (HSSB), T antigen, complexed to the core origin, started the unwinding of duplex DNA. At low concentrations of T antigen and in the presence of ATP, DNA polymerase alpha (pol alpha) stimulated the binding of T antigen to the core origin, while HSSB did not. This stimulation resulted in an increase in the subsequent unwinding reaction in the presence of HSSB. Primase alone did not affect the binding reaction and was not required for the stimulation by pol alpha. The stimulation required the hydrolysis of ATP and the AT tract domain of the core origin. Kinetic studies showed that while pol alpha stimulated the binding of T antigen to the core origin, it did not stabilize the complex. Pol alpha also stimulated the formation of the ATP-dependent T antigen-site I complex, a region that also contains an AT-rich sequence. These observations imply a regulatory role for pol alpha in the initiation of SV40 DNA replication.