Two phases of calcium mobilization were observed when aequorin-loaded human platelets, suspended in a nominally calcium-free medium containing 0.1 mM EGTA, were stimulated with collagen. The first phase coincided with platelet shape change, and the second phase corresponded to aggregation. On the other hand, only one [Ca2+]i peak was found in systems containing 1.0 mM Ca, or 1.0 or 2.0 mM EGTA. A novel antiplatelet compound alpha,alpha'-bis [3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, inhibited both [Ca2+]i peaks. It is suggested that inhibition of the mobilization of intraplatelet calcium stores as well as the blocking of transmembrane calcium flux may be responsible for the platelet aggregation-inhibitory action of this compound.