The DNA-dependent conversion of incorrect deoxynucleoside triphosphate precursors to monophosphates (turnover) by bacteriophage T4 DNA polymerase was determined using either poly(dA) x (dT) or poly(dG) x (dC) homopolymer templates. Competition between correct and incorrect triphosphates for incorporation into DNA, and the use of chain-terminating dideoxynucleoside triphosphates enabled us to determine the amount of turnover occurring at the end of each strand of the homopolymer duplex (e.g. amount of turnover of dATP occurring at the 3'-OH of poly(dG) and the 3'-OH of poly(dC)). These determinations suggest that nearest neighbor interactions between incoming dNTPs and the growing strand terminal residue play a major role in the occurrence of substitution errors during DNA synthesis in vitro byDNA polymerase. When considered together with existing evidence from studies of turnover (Gillin, F. D., and Nossal, N. G. (1976) J. Biol. Chem. 251, 5225-5232) and direct incorporation (Hall, Z. W., and Lehman, I. R. (1968) J. Mol. Biol. 36, 321-333) these results demonstrate that pyrimidine-pyrimidine and purine-purine as well as purine-pyrimidine oppositions have a role in error production at least during DNA replication in vitro. The implications of these results for the role of the "accessory" replication proteins in maintaining accuracy during the DNA biosynthetic process are discussed.