Transit of newly synthesized triacyl[3H]-glycerol through organelles of the secretory system leading to assembly into nascent very low density lipoproteins (VLDLs) or to cytoplasmic storage was investigated in chick hepatocytes. Cells in monolayer culture were pulse-labeled with [2-3H]glycerol, and after different periods of chase with unlabeled glycerol, the movement of triacyl[3H]glycerol through the endoplasmic reticulum (ER) and Golgi and the incorporation into nascent VLDL and cytoplasmic triacylglycerol-rich vesicles (TGRVs) were determined. Initially, newly synthesized triacyl[3H]glycerol is tightly associated with the ER. Movement from the ER of triacyl[3H]glycerol destined for cytoplasmic storage (as TGRVs) is extremely rapid and virtually complete within 8 min of chase. After 8 min of chase, triacyl[3H]glycerol lost from organelles of the secretory system was accounted for entirely as triacyl[3H]glycerol secreted as VLDL. Comparison of the rates of movement of triacyl[3H]glycerol, apolipoprotein B, apolipoprotein II, and apolipoprotein A-I through the ER and Golgi and of their secretion in nascent VLDL suggests that assembly of triacyglycerol with apolipoproteins occurs in the Golgi. Experiments with permeabilized hepatocytes supplemented with cytosol show that newly synthesized triacyl[3H]glycerol and [3H]phospholipid moves from the ER through the full-density range of Golgi elements and is dependent upon supplementary ATP.