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Specific predictions of the macromolecular crowding theory were examined on the volume of internally perfused barnacle muscle cells to analyze the mechanisms by which cells sense volume changes. The predictions tested were that isotonic replacement of large macromolecules by smaller ones should induce volume decreases proportional to the initial macromolecular concentration and size as well as to the magnitude of the concentration reduction. Results showed that isotonic replacement of proteins or polymers with sucrose induced volume reductions which agreed with the prediction.