The ubiquitous dinucleotide diadenosine tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. We have purified the pol alpha-associated Ap4A binding protein to homogeneity. The Ap4A binding protein is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. We have utilized [alpha-32P]8-N3-Ap4A to label the purified binding protein, and by cross-linking the photoaffinity label we have determined that Ap4A binds to the A1 subunit. No binding to the ligand is observed with the A2 subunit. Photoaffinity labeling is saturated with approximately 0.4 microM photolabel, with a half-maximal binding at 0.15 microM. The labeling is UV-dependent and is competed by both 8-N3-Ap4A and Ap4A. Photoaffinity labeling is not affected in the presence of dATP and dGTP and is reduced only in the presence of excess of ATP indicating the specificity of the protein for Ap4A. Of the diadenosine polyphosphates, Ap4A and Ap5A competed for binding, while Ap2A and Ap3A did not compete for binding. Further, the presence of at least one adenosine may be necessary since Ap4G competes but Gp4G does not compete for binding to the protein. Various methylene bisphosphonate and thiophosphate analogs of Ap4A were tested to see their effect on photoaffinity labeling with 8-N3-Ap4A. Significant differences were observed among the various analogs in their ability to prevent the photoaffinity labeling of the ligand to the binding protein.