In this study we have compared the intracellular itineraries of insulin and insulin-like growth factor-I (IGF-I) and their receptors subsequent to ligand internalization in rat fibroblasts. We found that the endocytic rate constant is approximately 3 times as high for insulin as for IGF-I. The dissociation of internalized ligand from its receptor was monitored by the ability of ligand-receptor complexes to precipitate in the presence of polyethylene glycol (PEG). Insulin loses its ability to precipitate with PEG more rapidly than IGF-I. After 60 min, less than 10% of the intracellular insulin remains PEG precipitable, whereas 44% of intracellular IGF-I stays PEG precipitable. Ligand degradation was determined by precipitation with trichloroacetic acid (TCA). Insulin degradation after internalization is more rapid compared with IGF-I degradation; after 2 h, 80% of intracellular IGF-I, in contrast to only 30% of intracellular insulin, remains intact. We measured retroendocytosis of insulin and IGF-I by assessing the amount of internalized ligand that was subsequently released from the cells. When analyzing released ligand by TCA precipitation, we found that 25% of insulin and 53% of IGF-I were TCA precipitable. After 120 min, only 16% of insulin and 43% of IGF-I remained intracellular. To provide insight into a possible mechanism that prevents IGF-I from being subjected to the same degree of degradation as insulin, we studied the effect of pH on IGF-I and insulin binding. We found IGF-I binding to be less sensitive to decreases in pH compared with insulin binding. Therefore, it is likely that after internalization, IGF-I does not dissociate from its intracellular receptor as easily as insulin in the acidifying endosome and, therefore, can return to the cell surface via a recycling receptor. In summary, we have observed distinct differences in the intracellular itineraries of IGF-I and insulin and their receptors. IGF-I internalization and degradation proceed less efficiently than insulin internalization, and IGF-I is preferentially targeted into a retroendocytotic pathway in contrast to insulin, which primarily undergoes lysosomal degradation. The differential effect of pH on ligand binding to these structurally related hormone receptors may account for the quantitatively distinct ligand trafficking events.