Studies were performed to determine whether formation of platelet aggregates itself, could cause an increase in cytosolic [Ca(2+)]([Ca(2+)](i)) which is independent of that resulting from the addition of agonists which induce aggregation. An increase in [Ca(2+)](i) did not coincide with aggregate formation when this response was dissociated from the addition of ADP or thrombin by delay either in initiating stirring or, for ADP, in adding fibrinogen. No increase in [Ca(2+)](i) occurred when aggregation was induced by addition of 1,2-dioctanoylglycerol or of ristocetin, or for chymotrypsin-treated platelets by addition of fibrinogen. The results demonstrate clearly that aggregate formation does not cause an increase in [Ca(2+)](i), and therefore exclude this possibility as an explanation for the discrepancies observed when [Ca(2+)](i) is measured, using aequorin and Fura2 as probes and as an underlying mechanism to account for contact-induced responses.