This paper presents a simple and efficient method for oligonucleotide-directed mutagenesis using vectors derived from single-stranded phage. This modification of our previously published procedure (Zoller and Smith, 1982) features the use of two primers, one of which is a standard M13 sequencing primer and the other is the mutagenic oligonucleotide. Both primers are simultaneously annealed to single-stranded template DNA, extended by DNA polymerase I (large fragment), and ligated together to form a mutant wild-type gapped heteroduplex. Escherichia coli is transformed directly with this DNA; the isolation of covalently closed circular DNA as in our previous report is not necessary. Mutants are identified by plaque lift hybridization using the mutagenic oligonucleotide as a probe. As an example of the method, a heptadecanucleotide was used to create a T----G transversion in the MATa gene of Saccharomyces cerevisiae cloned into the vector M13mp5. The efficiency of mutagenesis was approximately 50%. Production of the desired mutation was verified by DNA sequencing. The same procedure has been used without modification to create insertions of restriction sites as well as specific deletions of 500 bases.