In this study the exchange between 1H magnetization in “free” water (1Hf) and that in a pool with restricted motion (1Hr) was observed in tissues in vivo using NMR saturation transfer methods. Exchange between these two pools was demonstrated by a decrease in the steady-state magnetization and relaxation times of 1Hf with radiofrequency irradiation of 1Hr. The pseudo-first-order rate constant for the movement of magnetization from 1Hfto 1Hr was ∼ Is−1 in kidney and ∼3s−1 in skeletal muscle in vivo. Proton NMR imaging demonstrated that this exchange was tissue specific and generated a novel form of NMR image contrast. The extent of exchange between 1Hf and 1Hr as well as the topological correlation ofthe exchange with relaxation weighted images suggests that this pathway is a major determinant of the observed relaxation properties of water 1H in vivo. © 1989 Academic Press. Inc.