AbstractWhen the disulfide of 2‐mercaptoethanol (ESSE) is added to the medium of cultured Chinese hamster ovary (CHO) cells, a time and concentration dependent release of 2‐mercaptoethanol to the medium is observed. The reduction of ESSE to 2‐mercaptoethanol by cells is a saturable process, the rate being approximately 50 nmoles of 2‐mercaptoethanol per mg cell protein for an hour upon exposure to 250 μM ESSE. Reduction rate of ESSE by cells attached to a substratum is independent of glucose and insulin for periods up to 4 hours. However, in detached cells, swirled in suspension, addition of glucose and insulin is necessary in order to obtain a linear reduction rate of ESSE. The rate limiting enzyme in the sterol biosynthetic pathway, 3‐hydroxy‐3‐methyl‐glutaryl Coenzyme A reductase (E.C. 1.1.1.34), is inhibited by ESSE when isolated from CHO cells but total nonsaponifiable lipids synthesis from [2−14C]‐acetate in intact cells is not affected by ESSE at concentrations up to 500 μM. Cytosolic reduced glutathione can spontaneously exchange disulfide bonds with ESSE and thus prevent it from inhibiting the reductase. Cultured cells respond to ESSE administration by elevating their total and acid‐soluble glutathione levels. The use of ESSE as a perturbant of the GSH Statu