Mesophyll conductance, g_m, may pose significant limitations to photosynthesis and may be differentially affected by nutrition and genotype in Pinus radiata D. Don. Simultaneous measurements of gas exchange and chlorophyll fluorescence were made to determine g_m, using the constant J method (Harley, P.C., F. Loreto, G. Di Marco and T.D. Sharkey. 1992. Theoretical considerations when estimating the mesophyll conductance to CO₂ flux by analysis of the response of photosynthesis to CO₂. Plant Physiol. 98:1429–1436), in a fast- and a slow-growing clone of P. radiata grown in a greenhouse with a factorial combination of nitrogen (N) and phosphorus (P) supply. Values of g_m increased linearly with the rate of photosynthesis at saturating irradiance and ambient CO₂ concentration, A_sat (g_m = 0.020A_sat, r² = 0.25, P < 0.001) and with stomatal conductance to CO₂ transfer, g_s (g_m = 1.16g_s, r² = 0.14, P < 0.001). Values of g_m were greater than those of stomatal conductance, g_s, and the ratio (g_m/g_s) was not influenced by single or combined N and P additions or clone with a mean (±SE) value of 1.22 ± 0.06. Relative limitations to mesophyll conductance, L_m (16%) to photosynthesis, were generally greater than those imposed by stomata, L_s (13%). The mean (±SE) CO₂ concentration in the intercellular air spaces (C_i) was 53 ± 3 μmol mol⁻¹ lower than that in the atmosphere (C_a). Mean (±SE) CO₂ concentration in the chloroplasts (C_c) was 48 ± 2 μmol mol⁻¹ lower than C_i. Values of L_s, L_m and CO₂ diffusion gradients posed by g_s (C_a − C_i) and g_m (C_i − C_c) did not significantly differ with nutrient supply or clone. Mean values of V_cmax and J_max calculated on a C_c basis were 15.4% and 3.1% greater than those calculated on a C_i basis, which translated into different slopes of the J_max/V_cmax relationship (C_c basis: J_max = 2.11V_cmax, r² = 0.88, P < 0.001; C_i basis: J_max = 2.43V_cmax, r² = 0.86, P < 0.001). These results will be useful for correcting estimates of V_cmax and J_max used to characterize the biochemical properties of photosynthesis for P. radiata.