Gene A protein, the initiator protein of bacteriophage phi X174 DNA replication, cleaves synthetic single-stranded oligodeoxyribonucleotides at the same site as the corresponding sequence at the phi X origin. The results identify the recognition sequence within the decamer CAACTTGATA which is cleaved next to the G residue. Further requirements for cleavage of double-stranded DNA by the gene A protein are supercoiling and an A + T-rich domain adjacent to the recognition sequence.