Na+-dependent glycine transport was studied in reticulocyte-enriched fractions of blood obtained after massive bleeding of sheep. The activity is dependent on the sodium electrochemical potential and the membrane potential. The sodium chemical gradient was varied by changing external or internal Na+ and the membrane potential, by addition of valinomycin. Similar results were obtained with resealed reticulocyte ghosts. Under conditions optimal for sodium pumping (intracellular Na+ > 50 mM), ouabain inhibited glycine uptake prior to any measurable change in the cellular Na+. In these cells an electrogenic sodium pump is probably sufficiently active to contribute to the membrane potential. Na+-dependent glycine transport undergoes a marked decrease during long-term incubation at 37.degree. C. During this time the cells maintain their integrity and ATP content but undergo maturation as evidenced in the decrease in cells with reticulocyte morphology.