Pyrophosphate analogues, namely, pyrophosphorous, hypophosphoric, and hypophosphorous acids, were evaluated as inhibitors in elongation reactions and substrates in pyrophosphorolysis reaction catalyzed by HIV-1 reverse transcriptase and DNA polymerase I (the Klenow fragment). The substrate efficacy of hypophosphoric acid in pyrophosphorolysis reaction exceeded that of pyrophosphate for both enzymes by more than ten times. The product of the reaction was a dNTP analogue bearing a hypophosphate in the beta,gamma-position. Pyrophosphorous and hypophosphorous acids were neither inhibitors nor substrates for the enzymes. Kinetic parameters of the pyrophosphorolysis reaction catalyzed by HIV reverse transcriptase in the presence of hypophosphoric acid were evaluated. The dTMP analogue bearing a hypophosphate in the beta,gamma-position was synthesized and its substrate properties in elongation reaction catalyzed by HIV-1 reverse transcriptase were similar to those of natural dTTP. Hypophosphoric acid was capable of removing ddTMP, ddTMP(3'N3), and ddTMP(3'NH2) from the 3'-end of primers with an equal efficacy.