Cytoplasmic submembrane domains of the insulin-like growth factor I (IGF-1) receptor ranging from glycine 940 to proline 959 were investigated for their role in endocytosis of the IGF-1 ligand in rat pituitary GC cells stably expressing mutant human IGF-1 receptors. Replacement of each of three tyrosine residues within the juxtamembrane domain reduced the internalization rate (Ke) by 33% (943Y–>A), 47% (950Y–>A), and 41% (957Y–>A), respectively. Other substitutions within the submembrane region variably retarded receptor-mediated IGF-1 ligand endocytosis. Thus, each amino acid appears to independently contribute to the overall ligand-stimulated IGF-1 receptor internalization rate. The effect of receptor occupancy on IGF-1 receptor endocytosis was also tested. The rate of 125I-labeled IGF-1 internalization by transfectants overexpressing the wild type IGF-1 receptor was rapid but decreased markedly in the presence of increasing unlabeled IGF-1 (ED50 = 44,000 receptors/cell). Substitution by alanine for any of the three submembrane tyrosines increased the ED50 to > 56,000 receptors/cell, decreased the slope (Kic), and had a variable effect on Vmax in the presence of increasing unlabeled IGF-1. In contrast, 125I-labeled IGF-1 endocytosis by the mutant kinase-deficient 952STOP cells was slow and not further retarded by unlabeled IGF-1. These results suggest that ligand-mediated internalization of the human IGF-I is consistent with saturable interactions between the IGF-I receptor juxtamembrane region (glycine 940-tyrosine 957) and components of the endocytic apparatus.