Organoselenium compounds have important pharmacological properties. However, these compounds can cause toxicity, typically related to oxidation of endogenous thiols. The aim of this study was to investigate whether 2,2′-dithienyl diselenide (DTDS) has potential toxicity in vitro and in vivo. Therefore, sulfhydryl-containing enzyme activities, δ-aminolevulinic acid dehydratase (δ-ALA-D) and Na+–K+-ATPase were used to predict DTDS toxicity in rat brain homogenate in vitro. In in vivo experiments, a DTDS administration (50 or 100 mg kg−1, p.o.) to rats was performed and toxicological parameters were determined. DTDS inhibited δ-ALA-D (IC50 2 µm) and Na+–K+-ATPase (IC50 17 µm) activities in vitro. The inhibitory effect of DTDS on δ-ALA-D and Na+–K+-ATPase activities was restored by dithiothreitol. DTDS (5–25 µm) elicited a thiol oxidase-like activity. In vivo, DTDS (50 and 100 mg kg−1) caused systemic toxicity, evidenced by a decrease in water and food intakes and body weight gain, as well as the death of rats. DTDS at the dose of 100 mg kg−1 increased plasma alanine and aspartate aminotransferase activities and decreased urea levels. At 50 and 100 mg kg−1, it increased lipid peroxidation levels. At the highest dose, DTDS inhibited δ-ALA-D activity. By contrast, Na+–K+-ATPase activity and antioxidant defense were not altered in the brains of rats exposed to DTDS. In conclusion, interaction with the cisteinyl residues seems to mediate the inhibitory effect of DTDS on sulfhydryl-containing enzymes in vitro. In addition, high oral doses of DTDS induce toxicity in rats. Copyright © 2011 John Wiley & Sons, Ltd.