[3H]5'-N-Ethylcarboxamidoadenosine (NECA) labels both Al and A2 adenosine receptors in mammalian brain tissue. In the presence of 50 nM N'-cyclopentyladenosine (CPA), however, to block selectively the Al component of binding, [3H]NECA labels two binding components in striatal tissue. Such binding is dependent on the presence of Mg2+ ions and the removal of endogenous adenosine, using the catabolic enzyme, adenosine deaminase. The two binding components were observed in six mammalian species-rabbit, man, rat, mouse, guinea pig, and calf-with dissociation constants varying between 3.2 and 9.6 nM for the higher-affinity site and 54 and 232 nM for the lower-affinity site. In rat brain tissue, specific binding was reduced by the presence of 5'guanylyimidodiphosphate Gpp(NH)p, indicating the involvement of a G-protein. Gpp(NH)p effects were attributable to a conversion of a proportion of high-affinity to low-affinity components, as determined by saturation experiments. These results, together with the observation that Gpp(NH)p shifted the inhibition curve for the agonist, 2-chloro-adenosine(2-CADO), to the right indicate that the sites labeled by [3H]NECA in the presence of 50 nM CPA represent multiple affinity states rather than multiple receptor subtypes.Pharmacological examination of the higher-aff inity binding component using 2 nM [3H]NECA showed that binding was consistent with the labeling of an A2 adenosine receptor in all species. The order of activity for adenosine agonists was NECA > 2-CAD0 > R-phenylisopropyladenosine (R-PIA) > N'cyclohexyladenosine 2 CPA > > S-PIA. The activity ratios of the diastereomers of PIA (SIR) varied between 7.9 (mouse) and 14.5 (rat). The interaction of a series of 8-phenyl-substituted xanthines showed marked species differences with 1,3-dipropyl-8-(2-amin0-4-chloro) phenylxanthine (PACPX) being the most active at 14-15 nM in rabbit and man, 3-fold less active in calf, and 24-40-fold less active in guinea pig, mouse, and rat. A similar trend was seen for 8-phenyltheophylline (8-PT), 8-para-sulfo-phenyltheophylline (8-PST), and 1,3-diethyl-8-phenylxanthine (DPX), the relative order of activity of these adenosine antagonists being PACPX > 8-PT 2 DPX 2 8-PST. Caffeine and theophylline were much less active (1Cs0 > 10,000 nM), while the phosphodiesterase inhibitor 3-isobutyl-1 -methyl xanthine (IBMX I C5, , 2,257-4,410 nM) was 2-3 times more potent in man and calf than in the other species studied.