γ-Glutamylcysteine synthetase was isolated by means of a three-step method in highly active (specific activity, about 1400 units/mg) and apparently homogeneous form from rat erythrocytes. The enzyme has a molecular weight of about 100,000, and is composed of two subunits (M_r ∼ 75,000 and 25,000). The erythrocyte enzyme exhibits physicochemical, catalytic, and immunological properties that closely resemble those displayed by rat kidney γ-glutamylcysteine synthetase. The isolation procedure described here, which was also successfully applied to isolation of the enzyme from sheep erythrocytes, may be useful in exploring the properties of mutant forms of the enzyme.