We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site. This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand. After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha. A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids.