Quantification of leaf respiration is important for understanding plant physiology and ecosystem biogeochemical processes. Leaf respiration continues in the light (R ) but supposedly at a lower rate than in the dark (R ). However, there is no method for direct measurement of R and the available methods require nonphysiological measurement conditions. A method based on isotopic disequilibrium quantified R (R ) and mesophyll conductance of young and old fully expanded leaves of six species. R was compared to R determined by the Laisk method (R ) on the very same leaves with a minimum time lag. R and R were generally lower than R , and were not significantly affected by leaf ageing. R and R were positively correlated (r = 0.35), and both were positively correlated with R (r ≥ 0.6). R was systematically lower than R by 0.4 μmol m s . Using A/C instead of A/C curves, a higher photocompensation point Γ* (by 5 μmol mol ) was found but no influence on R estimates was observed. The results imply that the Laisk method underestimates actual R significantly, probably related to the measurement condition of low CO and irradiance. The isotopic disequilibrium method is useful for assessing responses of R to irradiance and CO , improving our mechanistic understanding of R .

 The results suggest that the Laisk method underestimated R L . The isotopic disequilibrium method is useful for assessing responses of R L to irradiance and CO 2 , improving our mechanistic understanding of R L .