The phosphate groups of nucleic acids are often the targets of mutagenic and carcinogenic alkylating agents. In order to study the effects of alkyl phosphotriester modification on the physical and biochemical properties of DNA, two diastereomeric ethyl phosphotriester modified decadeoxyribonucleotides, d-CpCpApApGp(Et)ApTpTpGpG isomer I and isomer II, were prepared. A phosphotriester synthetic procedure was used to specifically place ethyl triester groups with either an R or S configuration in the central dimer region of the decamer. Terminal deoxynucleotidyl transferase was used to add oligodeoxyadenylate tails to the 3' end of the decamers. The resulting oligomers were tested as templates for Escherichia coli DNA polymerase I with d-(pT)8pCpC as a primer. The rates and extents of polymerization directed by the modified templates were 25% (isomer I) and 50% (isomer II) less than those of an unmodified control template. Thus the presence of an ethyl triester group inhibits polymerization, the effectiveness of which is determined by the orientation of the ethyl group relative to the rest of the template backbone. These results suggest ethyl phosphotriester lesions could inhibit replication rates of cellular DNA.