A 37 kDa protein that binds to diadenosine tetraphosphate (Ap4A) was purified from human HeLa cells and identified as uracil DNA glycosylase/glyceraldehyde-3-phosphate dehydrogenase (UDG/GAPDH). Utilizing photoaffinity labeling with [α-32P]8N3-Ap4A, an Ap4A binding protein of 37 kDa was identified from HeLa cell nuclear extracts. The 37 kDa protein was purified to homogeneity and subjected to trypsin digestion followed by amino acid sequence analysis. Two peptide sequences were determined and both had complete identity with the amino acid sequence of the 37 kDa polypeptide of UDG/GAPDH. Purified UDG/GAPDH binds to Ap4A with the same affinity as the HeLa cell nuclear 37 kDa Ap4A binding protein, and monoclonal antibodies to UDG/GAPDH cross-react with the 37 kDa Ap4A binding protein. UDG/GAPDH has been previously demonstrated to have numerous nonglycolytic activities. The UDG function is involved in DNA repair by excision of uracil from DNA. GAPDH is a RNA binding protein and binds to tRNA and AU-rich RNA. The AU-rich RNA binding has been implicated in the regulation of AU-rich element dependent mRNA turnover and translation. The identification of UDG/GAPDH as an Ap4A binding protein may be physiologically relevant to the proposed role of Ap4A as a regulatory nucleotide in cell growth.