Binding sites for N-ethylcarboxamido[3H]adenosine (NECA) in rat brain membranes and cryostat sections were examined in relation to their sensitivities to displacement by unlabeled NECA and R(-)-phenylisopropyladenosine (R-PIA). In membrane fractions from cerebral cortex, cerebellum, hippocampus and striatum, nanomolar concentrations of these adenosine receptor agonists displaced [3H]NECA such that R-PIA was more effective than NECA, consistent with the presence of an A1-adenosine receptor. At concentrations of displacing agent higher than 1 microM, R-PIA was unable to displace [3H]NECA further in cerebral cortex, cerebellum and hippocampus. In striatum, a second R-PIA-sensitive component of [3H]NECA binding was evident which was more sensitive to NECA than to R-PIA, i.e. it showed the characteristics of an A2-adenosine receptor. In striatum, however, R-PIA was also unable to displace [3H]NECA binding completely. Similar results were obtained in quantitative autoradiographic studies. Preincubation of cryostat sections with N-ethylmaleimide (NEM) abolished both the A1- and R-PIA-insensitive binding components such that both NECA and R-PIA were able to displace [3H]NECA binding completely. The remaining sites showed IC50 values of 0.13 and 3.68 microM for NECA and R-PIA, respectively. These A2-like [3H]NECA binding sites had a highly specific distribution in the brain, being concentrated in the striatum, nucleus accumbens and olfactory tubercle. The results indicate the presence in brain tissue of at least 3 classes of [3H]NECA binding sites, an A1-site, an A2-site and a third, unidentified R-PIA-insensitive site.