The Sp diastereomer of thymidine 5'-O-(1-thiotriphosphate) was polymerized by avian myeloblastosis virus reverse transcriptase using poly(A) . d(pT)10 as template-primer. Degradation of the template poly(A) by alkaline hydrolysis and isolation by gel chromatography gave a single-stranded poly(d(p(S)T)), a polymer of thymidine 5'-phosphorothioate. To determine the configuration of the phosphorothioate internucleotide linkage, this material was degraded by snake venom phosphodiesterase. Comparison of the rates of degradation by snake venom phosphodiesterase of poly(d(p(S)T)) prepared by reverse transcriptase and DNA polymerase I showed them to be very similar. Since it has been established earlier than the latter enzyme produces polymers with phosphorothioate linkages of the Rp configuration (Burgers, P. M. J., and Eckstein, F. (1979) J. Biol. Chem. 254, 6889-6893), it is concluded that the polymer produces by reverse transcriptase has the same stereochemistry. Further proof for this assignment comes from comparison by 31P nmr of this polymer with the diastereomers of synthetic 5'-O-thymidyl 3'-O-thymidyl phosphorothioate. The chemical shift observed for the polymer was identical with that of the Rp isomer of 5'-O-thymidyl 3'-O-thymidyl phosphorothioate. Avian myeloblastosis virus reverse transcriptase therefore polymerizes deoxynucleoside 5'-triphosphates with inversion of configuration at the alpha-phosphorus. This result indicates that direct nucleophilic attack by the 3-hydroxyl group of the growing polymer on the alpha-phosphoryl group occurs without formation of a covalent enzyme intermediate.