Biochemical and morphological studies compared the ATP requirements for and the internalization routes of alpha 2-macroglobulin and insulin in H35 hepatoma cells. Cellular ATP concentrations were decreased more than 94% by 1 mM 2,4-dinitrophenol or 10 mM sodium azide, potassium cyanide, or oligomycin. ATP depletion decreased total cell-associated alpha 2-macroglobulin 70-90% by inhibiting binding 67-77% and receptor-mediated internalization 90-96%. Under the same conditions, insulin binding was decreased less than 10%, and endocytosis and intracellular accumulation were not affected. Quantitative electron microscopic analysis of the distribution of occupied receptors on the surface of control and treated cells was performed using colloidal gold-labeled alpha 2-macroglobulin or insulin. alpha 2-Macroglobulin concentrated in and was internalized almost exclusively by coated pits. Insulin was rarely associated with coated pits, but was found in and internalized by noncoated invaginations. ATP depletion did not affect receptor mobility or ligand-induced aggregation of either receptor. There was an increase in the amount of alpha 2-macroglobulin found in coated pit-like structures. The coat underlying pits in ATP-depleted cells was poorly defined and may account for the inability of coated pits to form and/or internalize. These results showed that receptor-mediated internalization via coated pits was ATP dependent, whereas internalization via pinocytotic invaginations was energy independent, which explained the difference in the ATP dependency of uptake for the two ligands. These observations suggested that autophosphorylation of the insulin receptor may not be involved in either the aggregation or internalization of the insulin-receptor complex, since ATP depletion did not affect either process. This study provided evidence that specialized mechanisms exist for the internalization of insulin which may be related to some of its intracellular effects.