Rabbit proximal colon epithelial cell apical membranes, which are known to contain receptors for insulin, were isolated by a Ca2+-precipitation technique. Binding assays with 125I-insulin-like growth factor I (IGF-I) revealed the presence of specific high-affinity binding sites, with 50% inhibition of binding observed at a concentration of 13.7 ng/ml IGF-I. In contrast, 50% inhibition of 125I-IGF-I binding was observed at an insulin concentration of 1.37 μg/ml, suggesting that 125I-IGF-I was not binding to insulin receptors present in this tissue. Cross-linking studies revealed an 125I-IGF-I binding subunit of relative molecular weight (M(r)) of 130,000 under reducing conditions on docecyl sulfate-polyacrylamide gel electrophoresis that was similar to the IGF-I binding subunit in human placental membranes (M(r) 140,000). Binding and cross-linking studies with 125I-insulin-like growth factor II (IGF-II), however, failed to reveal a specific receptor for this peptide in colon epithelial cell membranes. These results establish the coexistence of receptors for IGF-I and insulin, but not IGF-II, on rabbit proximal colon epithelial cell apical membranes and demonstrate that colon epithelial cells are capable of selective synthesis of various peptide hormone receptors.