The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5-2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro.