Abstract One-lane DNA sequencing by solvolysis in hot aqueous piperidine solutions, originally described for 5′- 32P-labeled DNA (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194–6200) , is extended to 3′-labeled fragments. A salt-free sample for electrophoresis can be obtained by using 1 m LiCl in the solvolysis mixture and removing this salt from the dried hydrolysate by washing with ethanol. Rate and distribution of DNA cleavage in hot aqueous piperidine, containing 0.3 m NaCl, are studied in dependence of temperature, solvent, amine concentration, and reaction time. An increase in temperature strongly accelerates overall DNA degradation, but leaves the distribution of cleavage essentially unchanged. When 50% aqueous ethanol is substituted for water as the reaction solvent, the overall cleavage is slower, and scission at G-sites is enhanced relative to cleavage at the other bases. A rise in the piperidine concentration strongly accelerates the reaction, except at very high amine concentration. Cleavage at A-, G-, and C-sites increases steadily with reaction time, while the T-cleavage observed takes place primarily at the very beginning of the solvolysis.