A novel A-Ci curve (net CO2 assimilation rate of a leaf -An- as a function of its intercellular CO2 concentration -Ci) analysis method (Plant, Cell & Environment 27, 137-153, 2004) was used to estimate the CO2 transfer conductance (gi) and the maximal carboxylation (Vcmax) and electron transport (Jmax) potentials of ageing, non-senescing Pseudotsuga menziesii leaves in relation to their nitrogen (N) content and protein and pigment composition. Both gi and the stomatal conductance (gsc) of leaves were closely coupled to Vcmax, Jmax and An with all variables decreasing with increasing leaf age. Consequently, both Ci and Cc (chloroplastic CO2 concentration) remained largely conserved through successive growing seasons. The N content of leaves, as well as the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other sodium dodecyl sulfate-soluble proteins, increased during the first three growing seasons, then stabilized or decreased only slightly afterwards. Thus, the age-related photosynthetic nitrogen use efficiency (PNUE) decline of leaves was not a consequence of decreased allocation of N towards Rubisco and other proteins involved in bioenergetics and light harvesting. Rather, loss of photosynthetic capacity was the result of the decreased activation state of Rubisco and proportional down-regulation of electron transport towards the photosynthetic carbon reduction (PCR) and photorespiratory (PCO) cycles in response to a reduction of CO2 supply to the chloroplasts' stroma. This study emphasizes the regulatory potential and homeostaticity of Cc- rather than photosynthetic metabolites or Ci- in relation to the commonly observed correlation between photosynthesis and gsc.