Amoebae were injected with a solution of 3H-choline, and samples were prepared for electron-microscopic radioautography at intervals between 15 min and 24 h thereafter. At the earliest interval, the rough endoplasmic reticulum was the most heavily labeled organelle. At subsequent intervals, the proportion of silver grains over the rough endoplasmic reticulum decreased rapidly, while that associated with other membranes increased. Most notably this involved a rapid rise in labeling of vacuoles, up to 1 h, and a more gradual increase in plasma membrane labeling up to 2 h. The results suggest that phospholipids are synthesized in the rough endoplasmic reticulum and are then transferred to other cellular membranes. A sequence of transfer steps suggested by the order of increases in labeling of different types of membranes is rough endoplasmic reticulum, smooth membranes and nuclear membranes, vacuoles, and the plasma membrane.