The development of a new preparation technique has allowed effects of fluid-substitution experiments on the tectorial membrane (t.m.) to be studied morphologically and not only, as previously, electrophysiologically. The organ of Corti and t.m. were examined in situ, unfixed and with in vivo-like conditions under light-microscopical control (differential-interference-contrast, magnification 400-800X). An irreversible shrinkage of the marginal and middle zones of the t.m. was observed under the influence of Na+ ions (substitution of endolymph with artificial perilymph). This shrinkage is also seen in specimens prepared for scanning electron microscopy after perfusion of scale media with perilymph. If endolymph is replaced by an isotonic solution of EDTA (ethylenediamine-tetraacetate) the t.m. can swell in its vestibulo-tympanal extent, e.g. from 39 to 72 μm. This swelling is reversible on addition of CaCl2. Although the size of the t.m. is not influenced to any great extent by changes in fluid osmolarity, reduction of H+ ion concentration leads to a minimal state of hydration of the protobril system at pH 4.3 (i.e. region of isolectric point). The mechanism of Na+ and Ca2+ effects are discussed. These results indicate that the subtectorial space is morphologically separated from the scala media proper by the marginal zone. © 1979.