The two isomers 3'-azido-3'-deoxythymidine 5'-triphosphate (erythro-AZT-TP) and 1-(3'-azido-2',3'-dideoxy-beta-D-xylofuranosyl)thymine 5'-triphosphate (threo-AZT-TP) were studied as inhibitors of the reverse transcriptase activity of avian myelobastosis virus. Kinetic analysis of the (rA)n X (dT)12-18 (a standard template primer complex of polyriboadenylate and oligodeoxythymidylate of indicated length)-directed reaction revealed that erythro-AZT-TP was a competitive inhibitor with respect to dTTP, whereas threo-AZT-TP was a noncompetitive inhibitor. The apparent Ki values, as calculated from Dixon plots, were 0.48 and 5.5 microM, respectively, compared with a Km value for dTTP of about 70 microM. These results indicate that erythro-AZT-TP had an approximately 150-times-higher affinity to the enzyme than dTTP had and that the avian myeloblastosis virus reverse transcriptase had different binding sites for the two isomers.