We have developed a general and rapid method for site-directed mutagenesis using primed amplification by the polymerase chain reaction. Starting from a double-stranded DNA template, this method requires only one single specific mutagenic primer and two universal sequencing primers flanking the region to be mutated further upstream and downstream, respectively. To test the method, two different mutants of the RNase T1-encoding gene have been constructed by this technique. Twelve sequenced mutant clones all showed the expected mutations without any wild-type background.