Freezing and thawing of leaves of herbaceous plants leads to damage when the freezing temperature falls below a certain tolerance limit, which depends on the plant species and state of acclimation. Such damage is expressed as an irreversible inhibition of photosynthesis observed after thawing. In frost-damaged leaves the capacity of photosynthetic reactions of the thylakoid membranes is impaired. Particularly, the water-oxidation system, photosystems II and I are inhibited. However, it appears that CO2 assimilation is more readily affected by freezing stress than the activity of the thylakoids. The inhibition of CO2 fixation seen in initial stages of damage seems to be independent of thylakoid inactivation. This can be shown by chlorophyll fluorescence analysis made simultaneously with measurement of CO2 assimilation. Fluorescence emission by leaves is strongly influenced by carbon assimilation activity, namely via the redox state of the photosystem II electron acceptor QA (QA-dependent quenching) and via energization of the thylakoid membranes depending on the transthylakoid proton gradient (energy-dependent quenching). Resolution of these components of fluorescence changes provides insight into alterations of the CO2 fixing capacity of the chloroplasts and properties of the thylakoids. The effects of freezing and thawing were studied in detail with isolated mesophyll protoplasts prepared from both non-hardened and cold-acclimated plants of Valerianella locusta L. Freezing damage was characterized by various parameters such as plasma membrane integrity, photosynthetic CO2 assimilation, chlorophyll fluorescence emission and activities of thylakoids isolated from the protoplasts. All tests indicated a substantially increased frost tolerance of protoplasts obtained from cold-acclimated as compared to non-hardened leaves. CO2 assimilation and related fluorescence changes were the most freezing-sensitive parameters in both types of protoplasts. Inactivation of CO2 assimilation was correlated neither to the disintegration of the plasma membrane nor to inactivation of the thylakoids. Experimental data indicate that freeze-thaw treatment affected the light-regulated enzymes of the carbon reduction cycle, such as fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase. Inhibition of light-activation of these enzymes may be based on altered properties of the chloroplast envelope.