The substrate properties of 3'-deoxythymidine 5'-triphosphate analogs prepared on the basis of 2,4-disubstituted 1,3-dioxolanes were investigated in reactions of the DNA synthesis catalyzed by various DNA polymerases. The 4'-triphosphates of (+/-)-cis-4-hydroxymethyl-2-(1-thyminylmethyl)-1,3-dioxolane and the corresponding (+/-)-trans-isomer were shown to be terminating substrates of terminal deoxynucleotidyl transferase. 4'-Triphosphate of (+/-)-cis-4-hydroxymethyl-2-(1-thyminylmethyl)- 1,3-dioxolane terminates the DNA synthesis catalyzed by HIV reverse transcriptase, whereas 2'-triphosphate of (+/-)-cis-2-hydromethyl-4-(1-thyminylmethyl)-1,3-dioxolane is a terminator in the DNA synthesis catalyzed by HIV reverse transcriptase and the Klenow fragment of DNA polymerase I.