AbstractBACKGROUND: Changes in chlorophyll fluorescence have often been advocated as a sensitive biomarker of plant stress, assuming that any kind of plant stress serious enough to affect plant growth will also affect photosynthesis. Glyphosate affects photosynthetic electron transport indirectly by inhibiting sink processes. The question is how fast this inhibition can be observed on CO2 assimilation and ultimately on chlorophyll fluorescence?RESULTS: Experiments measuring CO2 assimilation, conductance and chlorophyll fluorescence using four Kautsky curve parameters on barley (Hordeum vulgare L.) exposed to increasing doses of glyphosate showed a total cessation of CO2 fixation and conductance without significant changes in chlorophyll fluorescence. The decrease in CO2 fixation and conductance was significant 1 day after spraying and corresponded well to the decrease in biomass 5–7 days after spraying.CONCLUSION: A total cessation of CO2 assimilation can take place without affecting chlorophyll fluorescence. Hypotheses concerning what happens to the energy from the photosynthetic apparatus that is not used for CO2 assimilation are discussed. The results question the use of chlorophyll fluorescence as a universal indicator of stress on photosynthetic processes. Also, they demonstrate that changes in gas‐exchange parameters are more sensitive biomarkers for glyphosate toxicity compared with chlorophyll fluorescence. Copyright © 2010 Society of Chemical Industry