Longleaf pine (Pinus palustris Mill.) seedlings were exposed to two concentrations of atmospheric CO2 (365 or 720 μmol mol−1) and two levels of N (0·02 or 0·20 mg N g−1 soil yr−1) within open-top chambers for 20 months. Seedlings were adequately watered for 19 wk to ensure seedling establishment, after which two water-stress treatments (target values −0·5 or −1·5 MPa xylem pressure potential) were implemented. Fine-root samples were collected in July and November 1993, and in March and November 1994. Ectomycorrhizal and non-mycorrhizal short roots per unit length of fine root were quantified. The percentage of ectomycorrhizal short roots and numbers of ectomycorrhizas per unit root length were higher for seedlings grown with elevated CO2, low N and adequate water. Interactions among main treatment variables demonstrated higher percentages of ectomycorrhizal short roots, fine root length per seedling, and total numbers of ectomycorrhizas per seedling for plants grown with high CO2 (compared with ambient) or adequate water (compared with water stress) only under high N conditions. Increased fine-root length and ectomycorrhizal colonization under elevated CO2 resulted in higher (almost double) numbers of ectomycorrhizas per seedling at each sampling.