The role of Ca 2 + in the regulation of antidiuretic hormone(ADH)-induced water permeability of the apical membrane of the toad urinary bladder was examined. The effects of modifying Ca 2 + entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow (~bH2o) and short circuit current (Iso) were measured. In most experiments the bladders were treated with small amounts of Ag + (10 -7 mol/1) on the apical side. This treatment was used because previous experiments indicate that it markedly increases alkaliearth cation fluxes through an amiloride-insensitive cation channel in the apical membrane of the urinary bladder. Moreover, when Ca 2+ is the major cation in the apical solution of these Ag+-treated bladders, Isc is mostly due to Ca 2 + entry through the apical membrane. Ag + increased Iso and simultaneously inhibited ~H2o in bladders perfused with Ca 2 + solutions on the apical side. Addition of La 3+ to the apical solution reversed the stimulation of Isc and the inhibition of qsi~o produced by Ag + . When bladders were perfused with Ca 2 +-free solutions on the apical side, addition of Ag + did not inhibit ~bH~ o while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca 2 + solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (C1PheS-cAMP) the addition of Ag + did not inhibit ~H~o while it still increased Iso. Finally, addition of Ca 2 § to the apical solution of bladders not treated with Ag § reduced ~bn2o. These results taken together with other findings in the literature suggest' (1) Ca 2 + entry through the Ag+-treated amiloride-insensitive cation channel of the apical membrane inhibits ~H2o; (2) the effects of Ca 2 + entry are at a regulatory site that precedes the interaction of cAMP with the water channels; (3) it is also possible that Ca z + entry through the unmodified amiloride-insensitive cation channel may have some inhibitory effect on (~H20-Offprint requests to.