Perception of salt stress in plant cells induces a change in the free cytosolic Ca2+, [Ca2+]cyt, which transfers downstream reactions toward salt tolerance. Changes in cytosolic H+ concentration, [H+]cyt, are closely linked to the [Ca2+]cyt dynamics under various stress signals. In this study, salt-induced changes in [Ca2+]cyt, and [H+]cyt and vacuolar [H+] concentrations were monitored in single protoplasts of rice (Oryza sativa L. indica cvs. Pokkali and BRRI Dhan29) by fluorescence microscopy. Changes in cytosolic [Ca2+] and [H+] were detected by use of the fluorescent dyes acetoxy methyl ester of calcium-binding benzofuran and acetoxy methyl ester of 2′, 7′-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein, respectively, and for vacuolar pH, fluorescent 6-carboxyfluorescein and confocal microscopy were used. Addition of NaCl induced a higher increase in [Ca2+]cyt in the salt-tolerant cv. Pokkali than in the salt-sensitive cv. BRRI Dhan29. From inhibitor studies, we conclude that the internal stores appear to be the major source for [Ca2+]cyt increase in Pokkali, although the apoplast is more important in BRRI Dhan29. The [Ca2+]cyt measurements in rice also suggest that Na+ should be sensed inside the cytosol, before any increase in [Ca2+]cyt occurs. Moreover, our results with individual mesophyll protoplasts suggest that ionic stress causes an increase in [Ca2+]cyt and that osmotic stress sharply decreases [Ca2+]cyt in rice. The [pH]cyt was differently shifted in the two rice cultivars in response to salt stress and may be coupled to different activities of the H+-ATPases. The changes in vacuolar pH were correlated with the expressional analysis of rice vacuolar H+-ATPase in these two rice cultivars.