We describe a rapid and efficient megaprimer PCR procedure for site-directed mutagenesis that does not require any intermediate purification of DNA between the two rounds of PCR. This protocol is based on the design of forward and reverse flanking primers with significantly different melting temperatures ( T m). A megaprimer is synthesized in the first PCR reaction using a mutagenic primer, the low T m flanking primer and a low annealing temperature. The second PCR reaction is performed in the same tube as the first PCR and utilizes the high T m flanking primer, the megaprimer product of the first PCR and a high annealing temperature, which prevents priming by the low T m primer from the first PCR reaction. We have used this protocol with two different plasmids to produce cDNAs encoding seven distinct mutated proteins. We have observed an average mutagenesis efficiency of 82% in these experiments.