Summary Quantification of leaf respiration is of great importance for the understanding of plant physiology and ecosystem biogeochemical processes. Leaf respiration continues in light ( R L ) but supposedly at a lower rate compared to the dark ( R D ). Yet, there is no method for direct measurement of R L and most available methods require unphysiological measurement conditions. A method based on isotopic disequilibrium quantified R L ( R L 13C ) and mesophyll conductance of young and old fully-expanded leaves of six species compared R L 13C to R L values determined by the Laisk method ( R L Laisk ). R L 13C and R L Laisk were consistently lower than R D . Leaf ageing negatively affected photosynthetic performance, but had no significant effect on R L or R L / R D as determined by both methods. R L Laisk and R L 13C were measured successively on the same leaves and correlated positively ( r 2 =0.38), but average R L Laisk was 28% lower than R L13C . Using A / C c curves instead of A / C i curves, a higher photocompensation point Γ* (by 5 μmol mol -1 ) was found but the correction had no influence on R L Laisk estimates. 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 .