Hyperosmotic shrinkage (adding 300 microM sucrose to isotonic medium) stimulates lactate and ATP accumulation in rat but not human erythrocytes in which ATP pool was preliminary depleted. Inhibitors of Na(+)-K(+)-2Cl(-)-cotransport, Na+/H(+)- exchange and Na(+)- pump known to be activated by hypertonic medium had no influence on volume-induced effect. EGTA (1 microM), Ca(2+)-ATPase inhibitor vanadate (200 microM) and antagonist of calmodulin R24571 (10 microM) suppressed the stimulation of glycolysis by 80 and 30%, respectively. Addition of 1 microM Ca2+ and 1 mM Ca(2+)-ionophore A23187 in Ca2+ free medium stimulated glycolysis by 20% at isotonic conditions while additional hyperosmotic shrinkage resulted in a two-fold activation. It is suggested that shrinkage regulates activity of glycolytic enzymes through the mechanism of intracellular signaling involving Ca2+.