Treatment of 5'-end 32P-labeled ohgodeoxyribonucleotides with 0.4 M aqueous piperidinium formate, pH 2, at 37°C for 6 h, followed by treatment with I M aqueous piperidine at 90°C for 6 h, produces, after electrophoresis through 27% polyacrylamide sequencing gels, one-dimensional distributions of radioactivity from which the base sequences can be deduced. The order of intensities for the bands signaling the various bases is G > A > C > T. The spacing from a given band to the next higher band in the ladder was base characteristic, the order of band spacings being G > T ≥ A > C. In contrast to the one-cleavage one-lane DNA sequencing method reported earlier (B. J. B. Ambrose and R. C. Piess, 1985, Biochemistry 24, 6194-6200), which was based on treatment of end-labeled DNA with hot aqueous piperidine in the presence of sodium chloride, the present method produces a salt-free hydrolysate, thus minimizing electrophoretic irregularities in the fastest moving bands.