Specific receptors for insulin and the somatomedin peptides insulin-like growth factors I and II (IGF-I and IGF-II) have been characterized on three separate cloned strains of rat pituitary tumor cells (GH3, GH1, and GC). Binding of 125I-labeled peptides was time, temperature, and pH dependent for all three cell lines. Specific binding of [125I]insulin, which was extremely low in normal rat adenohypophyseal cells, was demonstrable for all three lines, with the Kd for the high affinity receptor ranging from 10(-10) to 4 X 10(-10) M/liter. A specific high affinity IGF-I receptor was also identified, with a Kd of approximately 10(-9) M/liter. IGF-II and insulin were, respectively, 10% and 1% as potent as IGF-I in competing for this receptor. When [125I]insulin and [125I]IGF-I were cross-linked to GH3 cells with disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both receptors were found to have an apparent mol wt greater than 300,000 in the unreduced state, with subunits of apparent mol wt 125,000 after reduction. A third discrete receptor, which bound [125I]IGF-II, was also identified on all three cell lines. IGF-I was only 10% as potent as IGF-II at displacing [125I]IGF-II, and insulin was virtually unreactive. When [125I]IGF-II was cross-linked to GH3 cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two receptors were identified. One had an apparent mol wt of 205,000 unreduced and 250,000 upon reduction, and presumably represents the type II receptor. Additionally, a band was observed at an apparent mol wt greater than 300,000 unreduced and 125,000 upon reduction, probably representing IGF-II binding to the IGF-I or insulin receptor. The presence of specific high affinity receptors for insulin, IGF-I, and IGF-II in these transformed cell lines is consistent with previous observations in normal rat and human pituitary cells and suggests a role for these peptides in the modulation of pituitary function.