A DNA polymerase is associated with the core of the so-called Dane particles. The probability that this is the hepatitis B viral DNA polymerase offers the possibility of preventing hepatitis B multiplication by selective inhibition of this enzyme. We have previously reported that trisodium phosphonoformate (PFA) inhibits Dane particle DNA polymerase. Fifteen compounds with structural similarity to PFA and pyrophosphate have now been tested for inhibition of hepatitis B virus DNA polymerase in an attempt to define the structural requirement for the inhibition. Active structures have two acid groups at close proximity of which at least one is a phosphono group. Phosphonoformate and hypophosphare were the two most active inhibitors. The Ki value for PFA was 7.2 microM when dTTP was used as variable substrate, and the mechanism of inhibition was non-competitive. Phosphonoformate caused rapid shut-off of the polymerase reaction, indicating that it might inhibit elongation. The efficient inhibition of hepatitis B virus DNA polymerase by PFA and its low toxicity suggest that it could be used to inhibit hepatitis B virus multiplication in vivo.