Abstract Up to three zones of γ-glutamyltransferase activity were present in 89 samples of human serum after agarose-gel electrophoresis at pH 8.6. Their mobilities relative to albumin were zero, 0.3–0.5, and 0.7–0.9. Incubation of human liver tissue in serum increased the activity of the zones with zero and 0.7–0.9 mobilities, and transiently, of the zone of intermediate mobility. More prolonged incubation caused the intermediate zone to decline, and produced new zones of mobility greater than that of albumin which were not seen in native sera. The mobility of partially-purified liver γ-glutamyltransferase incubated in serum or protein-free solutions was 0.7–0.8. The intermediate zone was not produced when liver tissue was incubated in protein-free solutions, nor with the purified enzyme in serum or protein-free solutions. The possible relevance of these observations to the electrophoretic patterns of γ-glutamyltransferase in pathological sera is discussed.