The effect of metabolic depletion on the maturation-associated loss of membrane functions has been studied by using sheep reticulocytes incubated in vitro at 37 degrees C for periods up to 41 hr. ATP was either maintained with glucose, adenosine plus inosine, or depleted with 2-deoxyglucose plus arsenate. Two membrane transport systems were studied: Na+-dependent glycine transport activity and the sodium pump, estimated from measurements of the number of [3H]ouabain binding sites per cell. Both transport systems were decreased during maturation. However, the decrease was much less in ATP-depleted cells compared to ATP-replete cells. It is concluded that the loss of certain functions during reticulocyte maturation is retarded by metabolic depletion.