Seven ectomycorrhizal fungi were screened by a soybean cell division bioassay to determine their potential for cytokinin synthesis. Fungal isolates of Rhizopogon roseolus (Corda) Hollos and Suillus punctipes (Peck) Sing, released significant quantities of cytokinin-like substances into the culture medium. In contrast, Cenococcum geophilum (Fr.), Hebeloma crustuliniforme (Bull. ex. St. Amans) Qual., Pisolithus tinctorius (Pers.) Coker and Couch, Suillus granulatus (L. ex. Fr.) O. Kuntze, and Thelephora terrestris (Ehrh.) Fr. failed to elicit a positive cytokinin response in the soybean bioassay. Purification of S. punctipes culture filtrates by cation exchange, organic solvent partitioning, and separation on Sephadex LH-20 revealed the presence of compounds with the same retention volume as zeatin and zeatin riboside. Greenhouse studies with loblolly pine (Pinus taeda L.) indicated that, together with improved plant growth and enhanced mineral nutrition, P. tinctorius mediated a significant effect on host cytokinin relations. Cytokinin activity of extracted needles increased 44 and 30% for 12- and 14-month-old seedings, respectively, following ectomycorrhizal establishment. Axenically grown seedlings colonized by 5. punctipes showed no alterations in growth, nutrition, or cytokinin concentration, compared to non-inoculated controls, even though mycorrhizal formation exceeded 30%. The ability of ectomycorrhizal fungi to affect host cytokinins was not dependent upon an ability to synthesize cytokinins in pure culture. Results are discussed in relation to improved mineral nutrition, enhanced root growth, and increased short-root proliferation in the ectomycorrhizal association.