Random mutagenesis of cloned DNA fragments can be an important tool for understanding genes and/or gene products. I report here a simple procedure for generating random mutations in any cloned DNA fragment in vitro. This method utilizes the polymerase chain reaction (PCR) to amplify chemically damaged single-stranded DNA containing the DNA fragment of interest. A mutagenized pool of plasmid DNA is produced by cloning the PCR product (containing chemically induced sequence heterogeneity) into a new vector. Therefore, extreme chemical treatments can be used that biologically inactivate the original vector. In this report, 10% of the plasmids contained a mutation in a 97-nucleotide DNA insert, giving a mutagenesis frequency of 10(-3)/base pair. Of 3000 yeast transformants screened, seven intron mutations were isolated that activate a cryptic 3' splice site, suggesting that 2% of the mutations could give rise to the desired phenotype. The seven mutations included transitions, transversions and single nucleotide deletions, demonstrating the well-balanced repertoire of nucleotide changes derived from this procedure.