A simple and efficient mutagenesis procedure is described which uses both the 3'----5' exonuclease and 5'----3' polymerase activities of T4 DNA polymerase. Different types of mutation-deletion, insertion, and substitution-can be introduced into the DNA in a single reaction. The technique uses recombinant M13 single-stranded DNA and two complementary DNA oligonucleotides to target and control the extent of deletions catalyzed by T4 DNA polymerase. The second oligonucleotide not only directs ligation, but also serves as a template for insertion or substitution of nucleotides by T4 polymerase. Mutant phages in a genetically pure form can be obtained at high efficiency, allowing their characterization directly by nucleotide sequencing without prior enrichment, plaque purification, and screening. We tested the versatility of this method by manipulating five regions of cDNA encoding the structural proteins of eastern equine encephalitis virus.