A1 adenosine receptors (A1ARs) are found in a number of tissues in the body where their physiological roles have been identified. In the cochlea, neither the existence of these receptors nor a physiological role of adenosine has been described previously. Membranes prepared from rat cochlea demonstrated high affinity and saturable binding to N6-2-(4-amino-3-[125I]iodophenyl)ethyladenosine ([125I]APNEA), an A1AR agonist, with maximum binding capacity and dissociation constant values being 40.5 +/- 0.5 fmol/mg protein and 1.28 +/- 0.03 nM, respectively. Adenosine analogues competed for [125I]APNEA binding sites with a rank order of potency characteristic of these sites being the A1AR. The [125I]APNEA binding was significantly reduced by pertussis toxin, indicating coupling of these receptors with the Gi and/or Go proteins in cochlear membranes. Photoaffinity labeling of the receptor protein with the A1AR agonist N6-2-(4-azido-3[125I]iodophenyl)ethyladenosine showed specific labeling of a 36-kDa receptor protein. Activation of the A1AR with R-phenylisopropyladenosine (R-PIA) led to inhibition of forskolin-stimulated adenylyl cyclase activity. Amplification of reverse-transcribed RNA derived from cochlear tissue by polymerase chain reaction (using primers for the bovine A1AR) yielded a 770-bp product that hybridized to an A1AR cDNA probe on Southern blots. These data indicate the presence of an inhibitory receptor in the peripheral auditory system, which may play an important role in modulating auditory functions.