We describe a new rapid method for random introduction of single-nucleotide (nt) substitutions into a small segment of cloned DNA. A DNA fragment containing a sequence to be mutagenized is inserted into a multiple cloning site sequence of a vector plasmid. The plasmid is linearized with two adjacent cuts (generating 5' and 3' protruding ends) and then synchronously and unidirectionally digested with exonuclease III (Exo III) so that the 3' termini generated are localized within the target region. A non-complementary alpha-thiophosphate nucleotide is misincorporated into the 3' terminus generated by Exo III. Since the nucleotide analogue is resistant to the 3'-5' exonuclease activity of DNA polymerase I, its misincorporation into the 3' termini is irreversible. Then, the single-stranded region is filled-in with four canonical nucleotides, and the plasmid is recircularized. This procedure was used to mutagenize a specific region of the rnpB gene of E. coli. By sequencing 72 randomly selected clones, we found that 27 clones (37.5%) had nucleotide substitutions distributed within the desired region of a 55-nt-long segment of the gene. The procedure is simple and is applicable to any DNA molecule.