Kinetic and pharmacological characteristics of Ca2+ fluxes across the saccular epithelium of trout were studied using a perfused isolated inner ear. Ca-45(2+) influx from the Ringer solution to the endolymph was 3-4 nmoles h(-1) mu(-1) endolymph, which corresponds to a global turnover rate of the endolymph calcium of 200% h(-1). Ca2+ entry into the proximal endolymph was faster than into the distal fluid. Net Ca2+ movement across the saccular epithelium depended on the direction and intensity of the chemical gradient of calcium between the Ringer solution and the endolymph. Increasing the calcium concentration in the Ringer solution up to 4.4 mmol l(-1) provoked an accumulation of Ca2+ in both proximal and distal endolymphs, and equilibrium was reached about 30 min after the beginning of perfusion. Perfusion with calcium-free Ringer partially emptied the proximal compartment of calcium, whereas the calcium levels in the distal endolymph did not vary during 70 min of perfusion. Verapamil (10(-5) mol l(-1)) and cyanide (CN, 10(-3) mol l(-1)) did not modify the accumulation of Ca2+ within the endolymph in the presence of a favourable calcium chemical gradient. Furthermore the relationship between Ca2+ net fluxes and the chemical calcium gradient across the saccular epithelium was linear, indicating a passive diffusional mechanism via a paracellular pathway. Similar relationships were found for Sr2+ fluxes across the saccular epithelium in the presence of positive chemical gradients (1, 2 and 4 mmol l(-1) Sr2+). In vivo experiments in which trout were intraperitoneously injected with CaCl2 solution confirmed the tight relationship between the calcium levels in plasma and endolymph (both proximal and distal). Sampling proximal and distal endolymphs in trout and turbot saccules revealed a decreasing proximo-distal calcium gradient in endolymph of both fish species. The present results strongly suggest that the endolymph is supplied with Ca2+ and Sr2+ via a paracellular pathway located in the proximal area of the saccular epithelium.