Suspensions of resting Escherichia coli were prepared from cell thawed and washed after frozen storage and from freshly harvested cells. Washing fluids, phosphate-free, were water or 0.05 ionic strength salt solutions. After washing, the cells were resuspended in initially phosphatefree solutions of sucrose, inorganic salts, or sodium citrate. Phosphate from the cells immediately began to accumulate in the suspending fluid. The rate of accumulation was examined briefly and the effect of solute concentration and molecular type on extent of accumulation was examined more extensively. In the presence of extracellular sucrose the extent of phosphate accumulation in a given time period was a monotonically increasing function of solute concentration. In contrast, during the same time period the use of any ionic extracellular solute tested resulted in a minimum accumulation at a common specific ionic strength. Evidence was developed supporting the hypothesis that the unidentified phosphate involved established an equilibrium across the cell membrane.