The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). Fluorescence titrations revealed noncompetitive inhibition of TNP-ADP binding by origin DNA, and noncompetitive inhibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able to bind the TNP nucleotide. From these titrations, we have measured a binding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral origin DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier. The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a preferred ligand over free T antigen in the double hexamer assembly reaction, preformed T antigen hexamers were incapable of forming double hexamers on the DNA. The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.