In various plant materials changes in turgor pressure, following hyper- or hypo-osmotic stress, were associated with the activation or inactivation of the plasma membrane H+-ATPase, respectively. To see if the turgor changes might indirectly influence H+-ATPase activity by regulating ion fluxes through plasma membrane, we investigated, in cultured cells of Arabidopsis thaliana (L.) Heynh., the early effects of hyper- and hypo-osmotic stress on Cl− fluxes in comparison, in the case of hyper-osmotic treatment, with its effect on net H+ extrusion. The results obtained showed that hyper-osmotic stress (200 mM mannitol) quickly reduced Cl− efflux (−70%) from cells preloaded with 36C1− for 18 h. This inhibiting effect was independent of the simultaneous mannitol-induced stimulation of Cl− influx and rapidly reversible after removal of the hyper-osmotic treatment. The inhibition of Cl− efflux was associated with a stimulation of net H+ extrusion, and these two effects showed the same dependence on the external mannitol concentration. Fusicoccin (FC, 20 µM), which stimulated H+ extrusion to about the same extent as 200 mM mannitol, did not affect Cl− efflux. When cells preloaded with 36C1− for 18 h in the presence of mannitol (from 25 up to 200 mM) were eluted in a mannitol-free medium an early and strong increase in Cl− efflux was found. The increase of Cl- efflux was already detectable for a small hypo-osmotic jump (25 mM), and was reduced (−50%) by the anion channel inhibitor A9C (300 µM). These results lead to exclude a direct causal relationship mediated by Em changes between the effects of osmoticum on Cl− efflux and net H+ extrusion, and favour the view that the changes in turgor pressure induced by hyper/hypo-osmotic stress may respectively induce an early inactivation/activation of stretch-sensitive anion channels.