The response of forest species to increasing atmospheric CO2, particularly under resource limitations, will require study in order to predict probable changes which may occur at the plant, community and ecosystem levels. Longleaf pine (Pinus palustris Mill.) seedlings were grown for 20 months at two levels of CO2 (365 and 720 μol mol1) in two levels of soil nitrogen (4 and 40 g m−2), and with two levels of soil moisture (–0·5 and –1·5 MPa xylem pressure potential). Leaf tissue was collected in the spring (12 months exposure) and autumn (20 months exposure) and examined using transmission electron microscopy (TEM) and light microscopy. During early spring, elevated CO2 magnified effects of N and water treatment on starch accumulation and in some cases contributed to altered organization of mesophyll chloroplasts. Disruption of chloroplast integrity was pronounced under elevated CO2, low N and water stress. In autumn, needles contained little starch; however, chloroplasts grown under high CO2 exhibited stress symptoms including increased plastoglobuli and shorter grana. A trend for reduced needle phloem cross-sectional area resulting from fewer sieve cells was also observed under elevated CO2. These results suggest that, in nature, longleaf pine seedlings may not benefit from a doubling of CO2, especially when soil resources are limiting.