We have previously found that the inhibition of strial Na+K+-ATPase by ethacrynic acid occurs at much higher concentrations (I50=5×10-3 M) than the inhibition of the endolymphatic potential (I50=1×10-5 M), which in itself is evidence against the concept that Na+K+-ATPase is a target of ethacrynic acid action in the stria vascularis. In order to check whether the stria vascularis accumulates the drug selectively (as is the case in tissue slices of kidney) we perfused the perilymphatic space with 14C-ethacrynic acid for 30 min. No accumulation of the drug above the concentration in the perfusion medium (5×10-5 to 5×10-3M) occurred in the stria vascularis. Since 30 min of perfusion produces maximal alterations of the endolymphatic potential at 1×10-4 M and above, and near maximal alterations at 5×10-5 M, Na+K+-ATPase can be excluded as a primary target of ethacrynic acid action upon the stria vascularis. No inhibition of Na+K+-ATPase by furosemide is detectable at 1×10-2 M. Adenylate cyclase of the stria vascularis is strongly inhibited by both ethacrynic acid and furosemide. In the case of ethacrynic acid the I50 (1×10-5 M) coincides with that of the endolymphatic potential. The I50 of adenylate cyclase with respect to furosemide is equally low (9×10-6 M), but the I50 of the endolymphatic potential is considerably higher (2×10-4 M). These results are consistent with the concept that interference with adenylate cyclase is a primary mode of action of ethacrynic acid and furosemide upon the stria vascularis. However, these data in themselves do not prove that this concept is valid under in vivo conditions. Both ethacrynic acid and furosemide are strong inhibitors of adenylate cyclase in the organ of Corti (I50= 8×10-6 and 3×10-6 M, respectively). Basal activities of adenylate cyclase in spiral ganglion, Reissner's membrane and macula sacculi are reported, as well as revised results for organ of Corti and stria vascularis.