Galactose-1-phosphate uridyl transferase (GALT), the deficient enzyme in classical galactosemia, was studied by Percoll-gradient age-fractionation of erythrocytes. For normal GALT, a rapid and substantial decrease in GALT activity and loss of most of two isozymes was found to occur in the reticulocyte fractions. The loss of activity was then followed by relative stabilization of both GALT-specific activity and microheterogeneity in mature and aging erythrocytes. When applied to the study of mutant GALT from galactosemic patients, the Percoll-gradient fractionation method permitted detection in the reticulocyte-enriched fractions of up to 5% of normal GALT-specific activity and an isoelectric focusing pattern essentially the same as that of normal GALT. Percoll-gradient fractionation of erythrocytes offers a simple and direct method to study characteristics of GALT activity and microheterogeneity in normal and galactosemic human erythrocytes.