The rat testis expresses high levels of A₁ adenosine receptors (A₁ AR) that couple to the inhibition of adenylyl cyclase activity. However, the physiological role of these receptors in the testis is not clear. Previous studies have documented a number of changes in the testis associated with the aging process. The goal of this study was to assess whether alteration in the expression and function of the testicular A₁ AR occurs in aging, using the Fischer 344 rats as an aging model. Quantitation of A₁ AR expression by radioligand binding of [³H]1,3-dipropyl-8-cyclopentylxanthine, an antagonist radioligand, indicates reductions in receptor number by 35 ± 13.3 and 53 ± 18.2% in 18- and 25-mo-old rats, respectively, compared with 3-mo-old rats. Similar reductions in A₁ AR expression were determined using Western blotting and receptor autoradiography. Quantitation of the G_i proteins using selective antibodies indicate age-dependent reductions in the levels of _i-1,2-, _i-3- and -subunits. Furthermore, the modulatory influences of guanosine 5'-O-(3-thiotriphosphate) on the binding of agonist and antagonist radioligands to the A₁ AR were substantially reduced. Northern blotting analysis of rat testicular poly(A)⁺ RNA indicates both a 3.4-kb transcript and a 5.6-kb transcript that hybridized to the canine A₁ AR cDNA probe. The levels of the 5.6-kb transcript were decreased by 24 ± 18 and 52 ± 3% in the 18- and 25-mo-old rats, respectively, compared with the 3-mo-old rats. These results indicate age-dependent deficits in the A₁ AR signal transduction pathway in the testes and predict concomitant reductions in the action of adenosine.