Abstract Temperature of the substrate significantly affected ectomycorrhizal development and survival and growth of Pinus taeda seedlings in aseptic culture. Isolates of Thelephora terrestris formed ectomycorrhizae on approximately 45 percent of the feeder roots at 14, 19, and 24°C and on 30 percent at 29°C; mycorrhizae were not formed at 34°. Poor survival and growth of these seedlings and noninoculated control seedlings at 34° were related to the absence of ectomycorrhizae. Isolates of Pisolithus tinctorius formed increasingly more ectomycorrhizae as temperatures increased; approximately 80 percent of the feeder roots were ectomycorrhizal at 34°. Ectomycorrhizal development and mycelial growth in pure culture were correlated at different temperatures for isolates of T. terrestris , but not for isolates of P. tinctorius . Mycelial growth in pure culture was maximum at 28°C for P. tinctorius , but ectomycorrhizal development was greatest at 34°. Relative seedling vigor was related to ectomycorrhizal development at all temperatures for T. terrestris . Relative seedling vigor increased with ectomycorrhizal development by P. tinctorius up to 29°, after which seedling vigor declined even though ectomycorrhizal development increased at 34°. Forest Sci. 16:424-431.