Replacement of alpha-, beta- and gamma-phosphate groups in 2'-deoxynucleoside 5'-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at alpha-, beta- and gamma-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that gamma-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the gamma-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase alpha. For dUTP analogues substituted at both gamma-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at gamma-phosphonate or C5 position. Meanwhile, C5-substituted beta, gamma-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2'-deoxythymidine 5'-[beta, gamma-(methylphosphinyl)methylphosphonyl]-alpha-phosphate and its 3'-azido-3'-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.