T4 DNA polymerase copolymerizes the SP isomers of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and 5'-O-(2-thiotriphosphate) with dTTP onto a poly(d(A-T) template in the presence of various metal ions. The corresponding RP diastereomers are inactive, independent of the metal ion used. The polymer resulting from the polymerization of the SP diastereomer of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and dTTP can be degraded by the 5' leads to 3' exonuclease activity of Escherichia coli DNA polymerase I and alkaline phosphatase (Brody, R. S., and Frey, P. A. (1981) Biochemistry 20, 1245-1252) to d(Tp(S)A). This material has the RP configuration as determined by comparison with the RP and SP diastereomers obtained by chemical synthesis and preparative separation by high performance liquid chromatography. This result indicates inversion of configuration at the alpha-phosphorus in the nucleotidyl transfer reaction and is compatible with the absence of a covalent enzyme intermediate.