Concurrent measurements of leaf gas exchange and on-line C-13 discrimination were used to evaluate the CO2 conductance to diffusion from the stomatal cavity to the sites of carboxylation within the chloroplast (internal conductance; g(i)). When photon irradiance was varied it appeared that g(i) and/or the discrimination accompanying carboxylation also varied. Despite this problem, g(i) was estimated for leaves of peach (Prunus persica), grapefruit (Citrus paradisi), lemon (C. limon) and macadamia (Macadamia integrifolia) at saturating photon irradiance. Estimates for leaves of C. paradisi, C. limon and M. integrifolia were considerably lower than those previously reported for well-nourished herbaceous plants and ranged from 1.1 to 2.2 mumol CO2 m-2 s-1 Pa-1, whilst P. persica had a mean value of 3-5 mumol CO2 m-2 s-1 Pa-1. At an ambient CO2 partial pressure of 33Pa, estimates of chloroplastic partial pressure of CO2 (C(c)) using measurements Of CO2 assimilation rate (A) and calculated values of g(i) and of partial pressure Of CO2 in the stomatal cavity (C(st)) were as low as 11.2 Pa for C. limon and as high as 17.8 Pa for peach. In vivo maximum rubisco activities (V(max)) were also determined from estimates of C(c). This calculation showed that for a g(i)ven leaf nitrogen concentration (area basis) C. para-disi and C. limon leaves had a lower V(max) than P. persica, with C. paradisi and C. limon estimated to have only 10% of leaf nitrogen present as rubisco. Therefore, low CO2 assimilation rates despite high leaf nitrogen concentrations in leaves of the evergreen species examined were explained not only by a low C(c) but also by a relatively low proportion of leaf nitrogen being used for photosynthesis. We also show that simple one-dimensional equations describing the relationship between leaf internal conductance from stomatal cavities to the sites of carboxylation and carbon isotope discrimination (DELTA) can lead to errors in the estimate of g(i). Potential effects of heterogeneity in stomatal aperture on carbon isotope discrimination may be particularly important and may lead to a dependence of g(i) upon CO2 assimilation rate. It is shown that for any concurrent measurement of A and DELTA, the estimate of C(c) is an overestimate of the correct photosynthetic capacity-weighted value, but this error is probably less than 1.0 Pa.