The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules.