The aim of this study is to investigate the inhibition characteristics of brain glutathione reductase (GSSGR) under reducing conditions. Sheep brain glutathione reductase (GSSGR, NAD(P)H: oxidized glutathione oxidoreductase, EC 1.6.4.2) was purified about 1 1000 fold with a method employing ammonium sulphate fractionation, heat denaturation, 2',5‘-ADP Sepharose 4B and Sepharose C16B chromatography steps. NADPH is an inhibitor of brain GSSGR. Sodium arsenite, which is a specific inhibitor of enzymes having two nascent -SH groups, had little inhibitory effect on GSSGR when incubated with the enzyme alone. When the enzyme was incubated with both NADPH and arsenite, inhibition was intensified.2-Mercaptoethanol and reduced glutathione enhanced the inhibition further; whereas oxidized glutathione protected the enzyme from inhibition. The reaction mechanism of the enzyme is discussed in the light of these findings.