Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) primes DNA synthesis in an in vitro system containing purified HeLa cell DNA polymerase alpha, deoxyadenosine triphosphate, and the double-stranded synthetic octadecamer template 5'-d-(G-G-A-G-G-C-T-T-T-T-T-T-G-G-A-G-G-C) (C-C-T-C-C-G-A-A-A-A-A-A-C-C-T-C-C-G)-d-5'; this octadecamer sequence is part of the origin region of DNA synthesis in simian virus 40. Ap4A is shown to be covalently linked to the first residue of the short deoxynucleotide chain synthesized under these experimental conditions. This template-primer system can initiate the new deoxynucleotide chain but cannot extend it beyond the A . T region.