The attachment of platelets to collagen-coated microtiter plates at 20 degrees C was inhibited strongly by depletion of metabolic energy or by addition of cytochalasins and was slightly inhibited by the intracellular Ca2+ chelator BAPTA. In keeping with their respective potencies as inhibitors of actin polymerization, cytochalasins D and H were the most potent inhibitors of adhesion, while cytochalasin B was the least potent. Energy depletion, cytochalasin D or, to a much lesser extent, BAPTA also inhibited platelet adhesion to collagen in a suspension assay system at 37 degrees C. Collagen-induced platelet cytosolic Ca2+ mobilization was inhibited up to 70% by cytochalasin D and abolished by energy depletion or BAPTA. Elevation of intracellular platelet calcium by treatment with ionomycin had little effect on platelet adhesion to collagen. We propose that rapid platelet spreading along collagen fibers is both energy- and actin-dependent and necessary to produce maximal adhesion needed to elicit Ca2+ mobilization required for subsequent responses.