Phosphoinositidase C activities sensitive to purine and pyrimidine nucleotides have been identified earlier in ampulla from Rana ridibunda semicircular canal. The aim of this study was to characterize the pharmacological properties of other P₂ receptors borne by this structure. A microassay was developed to measure the binding of [³⁵S]adenosine 5'-O-(2-thiodiphosphate) ([³⁵S]ADPS) to a few ampullas microdissected from frog semicircular canals. When determined at 4°C in the absence of divalent cations, [³⁵S]ADPS binding was saturable with incubation time and reversible after elimination of free radioligand. The dissociation kinetics were biphasic and comprised a major component that was rapidly reversible and a minor component that dissociated slowly. [³⁵S]ADPS binding was competitively inhibited by unlabeled ADPS with an apparent dissociation constant of 0.48 ± 0.09 µM and a Hill coefficient of 0.70 ± 0.06, and Scatchard analysis revealed a minor class of high-affinity binding sites (R_T1 = 52 ± 11 fmol [³⁵S]ADPS bound/ampulla and K_d1 = 0.15 ± 0.04 µM) and a major class of low-affinity binding sites (R_T2 = 436 ± 79 fmol [³⁵S]ADPS bound/ampulla and K_d2 = 2.0 ± 0.8 µM). The pattern of stereospecificity for recognition of unlabeled structural ATP analogs was ADPS  ,-methyleneadenosine 5'-triphosphate = ADP = adenosine 5'-O-(3-thiotriphosphate) > ATP = diadenosine tetraphosphate = AMP > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate  2-methylthioadenosine 5'-triphosphate > 2-desoxythymidine 5'-triphosphate = guanosine 5'-triphosphate = inosine-5'-triphosphate = xanthosine 5'-triphosphate = cytosine 5'-triphosphate = uridine 5'-triphosphate = uridine-5'-diphosphate, whereas cAMP and adenosine were devoid of activity. For antagonists, suramin revealed competitive inhibitor potencies, whereas reactive blue 2 and DIDS acted as pure noncompetitive inhibitors. Results suggest that the population of labeled receptors is heterogeneous and contains a low number of P_2Y-like receptors and a large number of P_2X-like receptors whose molecular subtypes and functions in endolymph homeostasis remain to be defined.