In the present study it is reported that [3H]NECA binds in a specific and saturable manner to membranes from the cerebral cortex of the rat. Scatchard analysis revealed two binding sites. The high affinity binding site (Kd 10.66 +/- 5 nM, Bmax 0.305 +/- 0.05 pmol/mg prot) was characterized by the following features: maximum binding at 25 degrees C, sensitivity to pretreatment with NEM and regulation by Gpp[NH]p, enhancing of binding in the presence of 1.0 mM MgCl2 and 1.5 mM CaCl2; the rank order of potency of several analogues of adenosine in competing with [3H]NECA for binding, was CHA greater than L-PIA greater than NECA greater than CADO. The low affinity binding site (Kd261.8 +/- 50 nM, Bmax 4.19 +/- 0.33 pmol/mg prot) showed maximum binding at 0 degrees C, insensitivity to pretreatment with NEM up to 1 mM and to regulation by Gpp[NH]p, and inhibition of binding in the presence of MgCl2 and CaCl2. The low affinity site was also present in membranes not pretreated with adenosine deaminase and, even in this condition, an IC50 of 188.5 +/- 36 nM for NECA and an IC50 of 4.35 +/- 0.20 microM for adenosine were found. It is concluded that the high affinity binding site is similar to the A1 adenosine receptors. The low affinity binding site is not classifiable among the A-type adenosine receptors, although it shows peculiar features shared both with the human platelet A2 receptor and the adenosine receptor formerly studied with [3H]adenosine in membranes from the brain of the rat; these results could reflect heterogeneity of adenosine receptors in central nervous system.