Effects of NaCl and sea-water salinity (SWS) on proline accumulation and activities of Δ 1-pyrroline-5-carboxylate reductase (P5CR), Δ 1-pyrroline-5-carboxylate synthetase (P5CS) and phospho enolpyruvate carboxylase (PEPC) in three aquatic macrophytes Hydrilla verticillata, Najas indica and N. gramenia, which differed in salinity tolerance, were studied in order to understand their possible role in providing resistance to salinity. All the three species showed enhancement in the contents of proline in response to the salinity treatments. The accumulation was more in response to SWS than in response to NaCl alone, probably because of the presence of Ca 2+ in SWS. The activity of P5CR was found to be increased in response to both NaCl and SWS treatments, more in the salt-tolerant N. gramenia than in the salt-sensitive H. verticillata. The proline content, however, did not increase in proportion to the increase in the activity of the enzyme. Unlike P5CR, the activity of P5CS, studied as γ-glutamyl kinase ( γ-GK, a constituent of P5CS), did not increase either in response to NaCl or SWS, but the salt-tolerant N. gramenia had much greater background (control level) activity of the enzyme than the salt-sensitive H. verticillata. The activity of PEPC was also much greater in N. gramenia, as well as in the moderately salt-tolerant N. indica, when compared to that in H. verticillata. Only H. verticillata showed an increase in the activity of the enzyme in response to the salinity treatments. The study indicated that the accumulation of proline in aquatic plants is most likely a consequence of ionic adjustments in which Ca 2+ plays an important role, and also that the P5CS may not be a rate limiting factor of proline biosynthesis in these plants in contrast to the reports available for terrestrial plants. The study further indicated that greater the P5CS activity, more the aquatic plants are tolerant to salinity, and that C 4 type of carbon metabolism may have some contribution to the salinity tolerance in these plants.