The Reissner's membrane (RM) separates in the mammalian cochlea the K⁺-rich endolymph from the Na⁺-rich perilymph. The patch-clamp technique was used to investigate the transport mechanisms in epithelial cells of RM freshly dissected from the guinea pig cochlea. This study shows a stretch-activated nonselective cationic channel (SA channel) with a linear current-voltage relationship (23 pS) highly selective for cations over anions [K⁺  Na⁺ (1) > Ba²⁺ (0.65) > Ca²⁺ (0.32)  Cl (0.14)] and activated by the intrapipette gradient pressure. The open probability-pressure relationship is best fitted by a Boltzmann distribution (half-maximal pressure = 37.8 mmHg, slope constant = 8.2 mmHg). SA channels exhibit a strong voltage dependency and are insensitive to internal Ca²⁺, ATP, and fenamates but are blocked by 1 µM GdCl₃ in the pipette. They are reversibly activated by in situ superfusion of the cell with hyposmotic solutions. Kinetic studies show that depolarization and mechanical or osmotic stretch modify the closed and open time constants probably by a different mechanism. These channels could participate in pressure-induced modifications of ionic permeability of the RM.