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Using fragments of human c-Ha-ras and mouse Ha-ras1 genes containing 8-hydroxyguanine (8-OH-G) in hypermutagenic codon 12, we analyzed the kinetics of DNA synthesis catalyzed by human Polκ. This translesion DNA polymerase, belonging to the Y-family, was found to be moderately inhibited by the presence of 8-OH-G on either mouse or human templates. From our previous results, inhibition of various polymerases by 8-OH-G increases in the following order: Polη < Polκ < Polβ < Polα, showing that major replicative and repair polymerases are more sensitive to this lesion than enzymes belonging to the Y-family. In the direct mutagenesis experiments, Polκ was found to be more mutagenic than Polη studied previously: it inserted dAMP more efficiently than dCMP opposite 8-OH-G. Polκ was also able to cause indirect mispair (‘action-at-a-distance’ mutagenesis), this effect being more distinct on mouse templates. Two adjacent 8-OH-G residues in codon 12 inhibited Polκ moderately and induced misincorporation of dAMP. However, this effect was not comparable to the strong relaxation of the enzyme specificity, observed previously in the case of Polη. Polκ catalyzed incorporation (and misincorporation of dAMP) much more efficiently on mouse templates, human DNA fragments being distinctly worse substrates. Interestingly, in direct mutagenesis systems, the preference for dAMP over dCMP was nearly the same on mouse and human templates.