5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-β-D-lyxofuranosy)thymine, 1-(2',3'-epithio-2',3'-dideoxy-β-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase β from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase ϵ from human placenta. K m values of ItTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5–2.5 times higher than K m value for dTTP. Furthermore, V max values for modified substrates are only 2–3 times lower than V max for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.