Studies on the nature and characterization of the specific binding of 125I-insulin to intact human platelets have been undertaken. Although under conditions of physiological buffer osmolality, a binding equilibrium was attained in 4-6 h at 4 degrees C, at higher temperatures (17 degrees C or 24 degrees C) equilibrium was reached only in the presence of very high buffer osmolality or 25 mM NaF. Under conditions of normal osmolality and in the absence of NaF, binding at 17 degrees C was not saturable. This phenomenon was specific for insulin or insulin-like hormones (the insulin-like growth factors) and did not occur with 125I-labelled growth hormone, ACTH or beta-endorphin. The non-saturable uptake of insulin appeared due to an energy-dependent specific sequestration or internalization of insulin by mechanisms probably involving the microtubule system. This study emphasizes the need to restrict this non-saturable uptake of insulin if one wishes to adequately study the platelet plasma membrane receptors for insulin. These data also indicate that there is a major interaction of insulin and insulin-like hormones with normal human platelets and support previous demonstrations of major insulin effects on platelet physiology in both normal and diabetic states.