Auranofin, a coordinated gold compound, inhibits in vitro DNA synthesis and displays in vivo antitumor activity. To understand the mechanisms of inhibition of DNA replication, we have examined the effects of auranofin and other gold complexes on the activities of purified cellular and herpesvirus-induced DNA polymerases, and on in situ DNA replication in permeabilized S phase KB cells. Evaluation of the data suggests the following conclusions. (1) The gold compounds varied in their abilities to inhibit DNA polymerase activities. DNA polymerase alpha was more sensitive to inhibition by gold compounds than DNA polymerase beta; (2) Inhibition of purified DNA polymerases by gold (I) compounds was noncompetitive with both DNA template and triphosphate substrates. Inhibition by SKF 101675, a gold (III) complex was competitive with DNA. (3) None of the gold compounds tested preferentially inhibited herpesvirus-induced DNA polymerases. (4) The gold complexes that inhibited in vitro DNA replication also inhibited in situ DNA synthesis. However, the potency and order of potency of the compounds varied between the in vitro and in situ systems. (5) Auranofin and other gold compounds inhibited the clonogenic capacity of KB cells in a concentration-dependent manner. The IC50 values measured in the clonogenic assay were significantly lower than those obtained from the in vitro and in situ DNA replication assays.