The species-specific inverse relation between atmospheric CO(2) concentration and stomatal frequency for many woody angiosperm species is being used increasingly with fossil leaves to reconstruct past atmospheric CO(2) levels. To extend our limited knowledge of the responsiveness of conifer needles to CO(2) fluctuations, the stomatal frequency response of four native North American conifer species (Tsuga heterophylla, Picea glauca, Picea mariana, and Larix laricina) to a range of historical CO(2) mixing ratios (290 to 370 ppmV) was analyzed. Because of the specific mode of leaf development and the subsequent stomatal patterning in conifer needles, the stomatal index of these species was not affected by CO(2). In contrast, a new measure of stomatal frequency, based on the number of stomata per millimeter of needle length, decreased significantly with increasing CO(2). For Tsuga heterophylla, the stomatal frequency response to CO(2) changes in the last century is validated through assessment of the influence of other biological and environmental variables. Because of their sensitive response to CO(2), combined with a high preservation capacity, fossil needles of Tsuga heterophylla, Picea glauca, P. mariana, and Larix laricina have great potential for detecting and quantifying past atmospheric CO(2) fluctuations.