The internal conductance from intercellular spaces to the sites of carboxylation (gi) has only been measured in a few tree species and not in conifers, despite the fact it may impose a large limitation on photosynthesis. The present study provides the first estimates of gi for a coniferous species, and examines variation in gi with height and its relationships to anatomical, biochemical and physiological traits. Measurements were made on upper and lower canopy current-year needles of 50-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). Needle thickness and specific leaf area decreased by 30% from the top to bottom of the canopy. These anatomical/morphological changes were accompanied by modest variation in allocation of N to chlorophyll and the chlorophyll a/b ratio. Allocation of N to Rubisco did not vary with height, but the ratio of Rubisco to chlorophyll did owing to the aforementioned changes in allocation to chlorophyll. The value of gi was estimated in one tree from concurrent measurements of carbon isotope discrimination and net photosynthesis. To examine the variation in gi among trees a second independent method based on day respiration and the difference between the chloroplastic and intercellular photocompensation points (photocompensation point method) was used. Estimates of gi obtained by the two methods agreed well with values varying between 0.14 and 0.20 mol m−2 s−1. It is estimated that gi limits photosynthesis by approximately 20% as compared to an approximately 30% stomatal limitation (under well-watered conditions). The value of gi scaled approximately with maximum rates of photosynthesis, which were significantly greater in upper canopy needles. Nevertheless, gi did not vary significantly with canopy height, owing to greater variability in gi than photosynthesis.