Ferrets have high-Na+ and low-K+ erythrocytes (113 and 5.4 mmol/l cell water) due to the lack of Na(+)-K+ pumps. Because ferret erythrocytes have a high capacity for Na(+)-K(+)-2Cl- cotransport, the present study was undertaken to evaluate cell volume-related changes in cotransport activity and its role in volume regulation. With cell shrinkage, Na(+)-K(+)-2Cl- cotransport is activated about twofold. A large bumetanide-insensitive Na+ uptake component that has not yet been described is found in shrunken erythrocytes. Its inhibition by amiloride (concn inhibiting 50% of maximal response = 12 microM) and the Na+ dependence of amiloride-sensitive extracellular pH changes measured in cells suspended in hypertonic unbuffered medium indicate that this flux represents Na+/H+ exchange. Shrinkage activation of both transporters follows a time lag of approximately 3 min and also requires normal levels of ATP. ATP depletion inhibits Na(+)-K(+)-2Cl- cotransport even at normal cell volume. Both transporters are partially inhibited by the protein kinase inhibitors staurosporine and K252a, and activators of protein kinases A and C do not affect transport. Okadaic acid inhibition of protein phosphatases activates Na(+)-K(+)-2Cl- cotransport to its maximal activity (same after shrinkage), but shrinkage and okadaic acid activation are not additive. In contrast, okadaic acid activates Na+/H+ exchange even in shrunken cells. These results indicate that cell shrinkage activates Na(+)-K(+)-2Cl- cotransport and Na+/H+ exchange probably by phosphorylation processes.