Transport and motility inhibitors have been used to classify different types of high-affinity cytochalasin B (CB) binding sites in 3T3 cells. The potency of phloretin and phlorizin as inhibitors of sugar uptake paralleled their effectiveness in displacing high-affinity bound CB from the cells, indicating that the two compounds compete with CB for binding to sites associated with sugar transport proteins. On the other hand, cytochalasins D and E, which did not inhibit sugar uptake, inhibited binding of CB to a portion of the high-affinity sites, most probably those associated with actin-containing cytoskeletal-contractile structures. A small amount of high-affinity CB binding remained in the presence of both phloretin and cytochalasin E, indicating that the cells have a third class of sites which is not related to either sugar transport or cell motility, When isolated membranes were examined, it was found that a fraction of each class of high-affinity CB binding sites were associated with the fraction. In contrast, only sites sensitive to cytochalasin D were recovered in a soluble extract of the cells.