We developed and applied an ecosystem-scale model that calculated leaf CO2 assimilation, stomatal conductance, chloroplast CO2 concentration and the carbon isotope composition of carbohydrate formed during photosynthesis separately for sunlit and shaded leaves within multiple canopy layers. The ecosystem photosynthesis model was validated by comparison to leaf-level gas exchange measurements and estimates of ecosystem-scale photosynthesis from eddy covariance measurements made in a coastal Douglas-fir forest on Vancouver Island. A good agreement was also observed between modelled and measured delta13C values of ecosystem-respired CO2 (deltaR). The modelled deltaR values showed strong responses to variation in photosynthetic photon flux density (PPFD), air temperature, vapour pressure deficit (VPD) and available soil moisture in a manner consistent with leaf-level studies of photosynthetic 13C discrimination. Sensitivity tests were conducted to evaluate the effect of (1) changes in the lag between the time of CO2 fixation and the conversion of organic matter back to CO2; (2) shifts in the proportion of autotrophic and heterotrophic respiration; (3) isotope fractionation during respiration; and (4) environmentally induced changes in mesophyll conductance, on modelled delta(R) values. Our results indicated that deltaR is a good proxy for canopy-level C(c)/C(a) and 13C discrimination during photosynthetic gas exchange, and therefore has several applications in ecosystem physiology.