Changes in the size, shape, and structure of the isolated tectorial membrane of the chick were measured in response to isosmotic changes in the ionic composition of the perfusion solution. Substitution of artificial perilymph (AP) for artificial endolymph (AE) caused a small (similar to 15%), slow (time constants tau similar to 12 min) shrinkage of the thickness of the tectorial membrane that was largely reversed on return to AE. Substitution of AP for AE alters not only the predominate cation (from K+ to Na+) but also the Ca2+ concentration(from < 7 mu mol/l to 2 mmol/l). Additional experiments were performed to separate effects of each of these changes. When a high-Na+, low-Ca2+ solution was substituted for a high-K+, low-Ca2+ solution (AE), the tectorial membrane swelled significantly, often to more than twice its original thickness (the largest swelling was 337%), with a slow time course (tau similar to 23 min). Addition of Ca2+ to either high-K+ or high-Na+ solutions caused rapid shrinkage of the tectorial membrane (tau similar to 2-3 min). Addition of the Ca2+ chelator EGTA caused rapid swelling (tau similar to 4 min). Large osmotic responses were only partially reversible and caused long-lasting changes. For example, long-duration solution changes that produced large, rapid osmotic responses early in an experiment tended to produce smaller and slower responses later in the experiment. In contrast, the small osmotic responses to short-duration solution changes were repeatable for tens of hours. Changes in ionic composition of the bath affected not only the thickness of the tectorial membrane but also its other dimensions. Responses were not generally isotropic; both the size and shape of the tectorial membrane generally changed. Consistent changes in microstructure accompanied the osmotic changes.