Recent evidence suggests that gamma-glutamyl transpeptidase may be involved in the transport of amino acids into the lactating mammary gland. The enzyme also is secreted in milk and is associated mainly with milk membranes. The objective of this study was to purify and characterize gamma-glutamyl transpeptidase from milk membranes. The enzyme has been purified from milk membranes by solubilization with Lubrol WX; treatment with acetone, deoxylcholate, and bromelain; and chromatography on ion exchange and molecular-sieving resins. gamma-Glutamyl transpeptidase was purified over 11,000-fold from milk. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicates that the enzyme is composed of two subunits with molecular weights of 57,000 and 25,500. Both subunits are glycoproteins and have been identified in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of whole milk membrane. Kinetic characteristics of the purified enzyme are similar to those determined for intact milk membranes and lactating mammary tissue indicating that the purified enzyme has not been modified functionally by the purification procedure.