• Morphological and anatomical characteristics of needles from different whorl levels were examined on Scots pines ( Pinus sylvestris ), grown for 4 yr under either ambient or elevated (ambient + 400 µ mol mol -1 ) CO 2 concentrations in open-top chambers.• Needle characteristics were studied using light microscopy, scanning electron microscopy and laser scanning confocal microscopy.• Under the elevated CO 2 treatment stomatal density was reduced on both adaxial and abaxial needle surfaces although the number of rows of stomata did not change significantly. Needle cross-sectional area increased by 10%; this was largely the result of an increase in needle thickness and, to a lesser extent, needle width. The increase in needle thickness was due to a large increase in mesophyll tissue. The relative area (i.e. proportion of the total area) of epidermis plus hypodermis, of resin canal, of xylem and of central cylinder decreased, whereas the relative area of needle phloem significantly increased.• The results suggest that a prolonged exposure to elevated CO 2 has an effect on needle structure, anatomical and stomatal characteristics of Scots pine needles.