Processing of circulating polypeptide hormones by vascular endothelial cells may be critical to hormone transport from the vascular lumen to tissue sites of action. The comparative processing of insulin-like growth factor II (IGF II) and insulin by cultured bovine aortic endothelial cells was examined. These cells possess high-affinity receptors for IGF II and insulin, as shown by competitive-binding studies. At 37°C, internalization determined by both resistance to an acid wash and electron microscopy was rapid with 50–70% of bound IGF II and insulin internalized at 60 min. Subsequently, between 70 and 75% of the internalized hormones were released from the cells within 60 min. Although most of both hormones were released intact, degradation of IGF II was greater than that of insulin by two- to threefold, as assessed by G-50 chromatography and trichloroacetic acid precipitability. Leupeptin, a specific lysosomal protease inhibitor, increased cell-associated 125I-labeled IGF II by 53.0 ± 6.0% and decreased degradation by 55%; however, it was without effect on 125I-labeled insulin. Chloroquine and monensin, which act at the lysosomes and at other sites, increased both cell-associated IGF II and insulin and decreased the degradation of both hormones. The increases in cell-associated 125I—IGF II produced by chloroquine (42.0 ± 7.4%) and monensin (78.3 ± 8.5%) were quantitatively similar to the decreases in IGF II degradation caused by the agents; however, the increase in cell-associated insulin was approximately threefold greater than could be accounted for simply by decreased insulin degradation. These results demonstrate two functional pathways for the processing of internalized hormones in the endothelial cell: a lysosome-targeted degradative pathway and a nonlysosomal pathway that may mediate the release of primarily intact hormone from the cells. IGF II processing and insulin processing by endothelial cells are different, due in part to greater channeling of IGF II, compared with insulin, into the degradative pathway. The capacity of endothelial cells to differentially process polypeptide hormones represents a potential mechanism that may modulate hormone delivery to tissue Sites Of action.