Twenty nine pyrophosphate analogues have been tested for their ability to inhibit herpes simplex virus type 1 (HSV-1) DNA polymerase activity. The structural requirements for active inhibitors and their mechanism of action have been investigated. Active compounds had two negatively charged groups at close proximity. The most active compounds contained free phosphono and/or carboxyl groups. Apart from phosphonoformate and phosphonoacetate, which were strong inhibitors, some other analogues, 2-phosphonopropionate, 2-phenylphosphonoacetate, oxalate, carbonyldiphosphate, methanehydroxy-diphosphonate and hypophosphate also inhibited the HSV-1 DNA polymerase activity. With the exception of hypophosphate the tested compounds inhibited the activity of calf thymus DNA polymerase alpha to a lesser extent than the activity of the HSV-1 DNA polymerase. All inhibitors gave linear uncompetitive inhibition of HSV-1 DNA polymerase with activated DNA as variable substrate. With the four deoxynucleoside triphosphates as variable substrates all inhibitors except hypophosphate gave linear non-competitive inhibition patterns. Hypophosphate caused a more competitive type of inhibition. The inhibition constants for the active compounds have been determined. Phosphonoformate, phosphonoacetate, methanehydroxydiphosphonate and hypophosphate also inhibited HSV-1 plaque formation in cell culture.