Mesophyll conductance, g(m), was estimated from measurements of stomatal conductance to carbon dioxide transfer, g(s), photosynthesis, A, and chlorophyll fluorescence for Year 0 (current-year) and Year 1 (1-year-old) fully sunlit leaves from short (2 m tall, 10-year-old) and tall (15 m tall, 120-year-old) Nothofagus solandrii var. cliffortiodes trees growing in adjacent stands. Rates of photosynthesis at saturating irradiance and ambient CO(2) partial pressure, A(satQ), were 25% lower and maximum rates of carboxylation, V(cmax), were 44% lower in Year 1 leaves compared with Year 0 leaves across both tree sizes. Although g(s) and g(m) were not significantly different between Year 0 and Year 1 leaves and g(s) was not significantly different between tree heights, g(m) was significantly (19%) lower for leaves on tall trees compared with leaves on short trees. Overall, V(cmax) was 60% higher when expressed on the basis of CO(2) partial pressure at the chloroplasts, C(c), compared with V(cmax) on the basis of intercellular CO(2) partial pressure, C(i), but this varied with leaf age and tree size. To interpret the relative stomatal and mesophyll limitations to photosynthesis, we used a model of carbon isotopic composition for whole leaves incorporating g(m) effects to generate a surface of 'operating values' of A over the growing season for all leaf classes. Our analysis showed that A was slightly higher for leaves on short compared with tall trees, but lower g(m) apparently reduced actual A substantially compared with A(satQ). Our findings showed that lower rates of photosynthesis in Year 1 leaves compared with Year 0 leaves were attributable more to increased biochemical limitation to photosynthesis in Year 1 leaves than differences in g(m). However, lower A in leaves on tall trees compared with those on short trees could be attributed in part to lower g(m) and higher stomatal, L(s), and mesophyll, L(m), limitations to photosynthesis, consistent with steeper hydraulic gradients in tall trees.