AbstractThe binding of cytochalasin B (CB) to human platelets and to isolated platelet cytosol and membranes has been analyzed with [3H]CB. High‐ and low affinity classes of saturable binding sites were associated with intact platelets. Binding at very low concentrations of CB (i.e., high‐affinity binding) was partially prevented by 100 mM D‐galatose or D‐glucose and to a much lesser extent by L‐glucose. Binding to platelet cytosol also involved two classes of sites with affinities and capacities similar to those observed with the whole cells. None of this binding, however, was affected by 100 mM D‐galactose. Saturable binding to platelet membranes occurred at sites with a uniform binding affinity. Approximately 52% of this binding was prevented by 1 M D‐galactose and another 15% by cytochalasin E (CE). We hypothesize that binding in the cytosol is to monomeric (low‐affinity) and polymerized (high‐affinity) actin, whereas membrane binding (high‐affinity only) occurs primarily at sites involved with galactose transport.