The semicircular canals of the vestibular labyrinth of the dogfish shark, Squalus acanthias, may serve as a simple in vitro system for the study of the ionic transport mechanisms involved in endolymph formation. Electron microscopy showed that the epithelium was made up of at least three distinct cell types divided into separate regions running the length of the canals. Secretion of endolymph was studied in isolated canals by the split droplet method; when the lumens of the canals were filled with shark Ringer a potassium-rich fluid was secreted into the lumen at a rate of 0.34 microliter.cm-2.min-1. The K concentration of the secreted fluid averaged 112 mM; the calculated rate of K secretion was 2.3 mumol.cm-2.h-1, comparable to recent measurements in mammalian utricle. Fluid secretion was dependent on active transport and was inhibited by ouabain, bumetanide, or methazolamide in the external bathing solution. Fluid secretion was unaffected by the K channel blocker, Ba, in the luminal droplet or by the adenylate cyclase stimulator, forskolin, in the external bathing solution. For electrophysiological analysis, isolated canals were perfused in a chamber designed for voltage/current clamping; an axial wire was inserted into the canal lumen and constant-current pulses were passed to determine tissue resistance. When the luminal fluid was high-K Ringer, transepithelial potential difference was -1.1 mV (lumen negative) and resistance 37 omega.cm2. Dilution and bi-ionic potential measurements showed that the epithelium of the canals exhibited only slight cation selectivity. These results are consistent with a model for endolymph secretion involving cotransport secondary to the ion gradients created by the Na+-K+-ATPase.