Water stress by polyethylene glycol (PEG)-6000 solution (Ψs=0.2 MPa,stress time:48 h,rehydration time:48 h) was performed in leaves of two alfalfa cultivar (Long-Dong and Algonquin) seedlings.Gas exchange parameters,chlorophyll fluorescence parameters,activity of antioxidant enzyme and photosynthetic pigment concentrations were measured to investigate the available photosynthetic and antioxidant enzyme response to variable water conditions as well as stomatal and non-stomatal limitations to photosynthesis.The results showed that non-stomatal limitations were responsible for the reduction of photosynthesis during water stress.At the beginning of water stress (12 h),water was lost and then the stomata closed rapidly,which resulted in a decrease of transpiration,net photosynthesis and CO2 diffusion.Therefore,when intercellular CO2 concentration and carboxylation efficiency decrease,water use efficiency and value of stomatal limitation would increase.However,the decline of net photosynthetic rate was faster than transpiration rate.At the same time,the maximal photochemical efficiency,potential activity of PSII reaction center and photochemical quenching of chlorophyll fluorescence declined significantly,the activity of antioxidant enzyme increased rapidly and the photosynthetic pigment concentrations changed slightly.The results also indicated that,at the initial period of stress,neither oxidative stress nor membrane lipid peroxidation was induced,nor were photosynthetic structures damaged,but photosynthetic functions were partly inhibited.Therefore,the stomatal limitation and non-stomatal limitations had the same responsibility for the reduction of photosynthesis.At the mid-late stage of water stress,net photosynthetic rate,stomatal conductance,maximal photochemical efficiency,potential activity of PSII reaction center and photochemical quenching of chlorophyll fluorescence decreased linearly with the decline of the relative water content.And the relative electron transport rate,the effective quantum yield of PSII photochemistry and photosynthetic pigment concentrations declined continually.The activity of antioxidant enzymes maintained at a higher level but malondialdehyde accumulated gradually with prolonging of water stress.Simultaneously,the non-photochemical quenching of chlorophyll fluorescence increased obviously after water stress for 24 h.The remarkable decline of light saturated point of photosynthetic electron transport,that is,the initial point of photo-inhibition,was observed in advance.Therefore,non-stomatal limitations dominated the changes of a series of physiological and biochemical reactions during mid-late period of water stress.After 48 h rehydration,all the parameters except intercellular CO2 concentration in Long-Dong recovered obviously but incompletely,which resulted from severe oxidative injury and photo-inhibition induced by water stress even though photo-protection was triggered during water stress in alfalfa leaves.Alfalfa seedlings were sensitive to water stress and there were certain differences between cultivars.