The effects of intercalating agents on the fidelity of DNA synthesis in vitro have been investigated. The accuracy of DNA synthesis with Escherichia coli DNA polymerase I with both the poly[d(A-T)] and poly[d(G-C)] templates is decreased in the presence of the intercalating agents proflavin, ethidium bromide, acridine orange, ICR-170, and ICR-191. Nearest neighbor analyses of the product of the reaction indicate that two different types of misincorporations occur in the presence of intercalating agents, frameshifts, and single-base substitutions. With alternating polynucleotide templates, frameshifts involving pyrimidines are the most frequent change in sequence observed. Overall, frameshift misincorporations occur with frequencies of one complementary pyrimidine for each intercalated site and one noncomplementary pyrimidine for each 150 sites. From analysis of nearest neighbor frequencies in the product, it is inferred that the intercalating agents interact specifically with pyrimidine (3' leads to 5') purine sequences. An analysis of ratios of correct nucleotide incorporations as a function of intercalator concentration indicates that frameshifts are predominantly additions; however, one cannot rule out infrequent deletions. Base substitutions in the presence of intercalators occur less frequently than frameshifts. From the results of reaction kinetics and nearest neighbor frequencies, it is concluded that the noncomplementary nucleotides are incorporated in phosphodiester linkage and are present as single-base substitutions. Taken together, the results of these studies suggest at least two different modes of action for intercalating agents on the accuracy of DNA synthesis: one leading to frameshift misincorporations and the other leading to single-base substitutions.