Seven methods, including measurements of photosynthesis (A) and stomatal conductance (gs), carbon isotope discrimination, ecosystem CO2 and water vapour exchange using eddy covariance and the use of a multilayer canopy model and ecosystem Keeling plots, were employed to derive estimates of intercellular CO2 concentration (Ci) across a range of spatial and temporal scales in a low productivity rain forest ecosystem dominated by the conifer Dacrydium cupressinum Lamb. in New Zealand. Estimates of shoot and canopy Ci across temporal scales ranging from minutes to years were remarkably similar (range of 274–294 µmol mol−1). The gradual increase in shoot Ci with depth in the canopy was more likely attributable to decreases in A resulting from lower irradiance (Q) than to increases in gs due to changes in air saturation deficit (D). The lack of marked vertical gradients in A and gs at saturating Q through the canopy and the low seasonal variability in environmental conditions contributed to the efficacy of scaling Ci. However, the canopy Ci estimate calculated from the carbon isotope composition of respired ecosystem CO2 (δ 13CR; 236 µmol mol−1) was much lower than other estimates of canopy Ci. Partitioning δ 13CR into four components (soil, roots, litter and foliage) indicated root respiration as the dominant (> 50%) contributor to δ 13CR. Variable time lags and differences in isotopic composition during photosynthesis and respiration make the direct estimation of canopy Ci from δ 13CR problematic.