A procedure for determination of the intracellular water content of cells using a single, nonlabeled solute as an interstitial space marker is proposed. Sodium thiosulfate, which can be accurately assayed by a tritrimetric method, is found to be a good compound for this purpose. Cells are recovered both by filtration and centrifugation; the two techniques gave the same value for internal water, i.e., 650 mg of H2O/g of wet matter for Corynebacterium melassecola and 390 mg of H2O/g of wet matter for Penicillium roquefortii spores. The methodology of data handling, based on a regression technique, is also described. It allows one to obtain very reliable results and should be useful for any marker.