Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30‐min incorporation of [3H]thymidine into DNA following a 30‐min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4‐benzenetriol, hydroquinone, andp‐benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell‐free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [3H]thymidine triphosphate into DNA up to 24 μMbut 1,2,4‐benzenetriol and p‐benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4‐dithiothreitol (1 mM) in the cell‐free assay system. Furthermore, when DNA polymerase a, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase I, which does not require an SH group for its catalytic activity, p‐benzoquinone and 1,2,4‐benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply thatp‐benzoquinone and 1,2,4‐benzenetriol inhibited DNA polymerase a, consequently resulting in inhibition of DNA synthesis in both cellular and cell‐free DNA synthetic systems. The present study identifies catechol, hydroquinone, p‐benzoquinone, and 1,2,4‐benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause.