The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37 degrees C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37 degrees C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37 degrees C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.