Although agriculture focuses on row crops in the Midwestern US, chestnut (Castanea) would be a possible multipurpose agroforestry crop in erodible locations within this region. As ectomycorrhizal (ECM) colonization is often crucial for tree establishment and production, we addressed the importance of ECM colonization on chestnut performance by assessing: (1) natural ECM colonization in an established chestnut orchard, and (2) the effect of ECM inoculation on seedling establishment and drought response in a greenhouse. We selected 50 Chinese American hybrid chestnuts trees in a commercial orchard and assessed their level of mycorrhizal colonization in relation to distance from an adjacent forest, the source of inoculum. In the green house, we grew 80 seedlings from this orchard with and without mycorrhizal inoculation. Half were harvested to assess inoculation success and biomass; the remainder were subjected to experimental water stress which was evaluated with chlorophyll fluorescence using Fv/Fm values. In the orchard planted with uninoculated plants, ECM colonization decreased with distance from adjacent oak forest (P = 0.015), the putative ECM source, with an average of 29% root tips colonized. ECM inoculation increased seedling aboveground biomass by 16.4% in the greenhouse and generated 1.14 times more, but smaller stomata on leaves. Water stress in ECM inoculated seedlings also recovered faster from an experimental drought having significantly higher Fv/Fm value (P = 0.004) than uninoculated seedlings. Our study clearly indicates that ectomycorrhizal colonization can help chestnut trees in their early growth and stress tolerance and should be included into field plantings to minimize dependence on agricultural amendments. [ABSTRACT FROM AUTHOR]