The in vitro accuracy of DNA replication has been investigated through the measurement of the frequency with which noncomplementary nucleotides were incorporated during polynucleotide replication. The effect of beta-propiolactone treatment of deoxynucleotide templates, ribopolynucleotide templates, and the DNA polymerase from avian myeloblastosis virus was determined. Treatment of the deoxynucleotide template, poly(dA) (see article) oligo(dT) 12-18, by beta-propiolactone resulted in an increased frequency of noncomplementary nucleotide incorporation during DNA polymerization. Carcinogen treatment of the ribonucleotide templates, poly(rA) (see article) oligo(dT) 12-18, and poly(rC) (see article) oligo(dG) 12-18, and carcinogen treatment of avian myeloblastosis virus DNA polymerase did not alter the frequency of noncomplementary nucleotide incorporation. This suggested that carcinogen-induced error incorporation during DNA synthesis was restricted solely to the treatment of a deoxynucleotide template.