Selenite has been shown previously to inhibit cellular RNA synthesis. Based upon our previous observation that selenite inhibits purified RNA polymerase only in the presence of a sulfhydryl compound (Frenkel et al., Mol Pharmacol 31: 112-116, 1987), we hypothesized that the inhibition of cellular RNA synthesis by selenite involves endogenous sulfhydryl compounds. We found that depletion of cells of endogenous sulfhydryl compounds, by exposure to diethylmaleate (DEM), virtually eliminated the inhibitory effect of a 1-hr exposure of cells to selenite. This inhibition was restored to normal or higher levels when the selenite was reacted with glutathione or cysteamine prior to addition to the DEM-treated cells. RNA synthesis in DEM-treated cells was inhibited after a 4-hr exposure to higher concentrations of selenite. In contrast to the effect of DEM, specific depletion of the cells of glutathione, by exposure to buthionine sulfoximine, had no effect on the inhibition of RNA synthesis by selenite. These results demonstrate the involvement of endogenous cellular sulfhydryl compounds in the inhibition of RNA synthesis by selenite, but indicate that glutathione, in particular, is not involved in this inhibition.