Binding of [3H]-adenosine to a microsomal fraction derived from media strips of hog carotid arteries was studied by a centrifugation technique. Specific binding was very rapid, readily and completely reversible, and temperature dependent. Under the chosen conditions of incubation, at 4°C and in the presence of deoxycoformycin, about 95% of the bound radioactivity was chromatographically identified as adenosine. Scatchard analysis revealed a small number (1.7 pmol/mg protein) of high affinity (KD = 0.23 µmol/l) and a large number (21.6 pmol/mg protein) of low affinity binding sites (KD = 4.3 µmol/l). 5’-Phosphorylated adenosine compounds, 5’-deoxyadenosine, 2-chloroadenosine, and adenosine-5’-N-ethylcarboxamide, all derivatives of adenosine with pronounced vasodilatory properties, were most potent in displacing [3H]-adenosine from its binding sites with IC50 values ranging from 1.8 to 14.3 µmol/l. However, 2-chloroadenosine and (–)N6-phenylisopropyladenosine were weaker competitors of binding than adenosine, which disagrees with the pharmacological findings. Moreover, the IC50 of aminophylline was much higher than the concentration required for half-maximal inhibition of the biological actions of adenosine. This partial lack of specificity may be due to nonreceptor binding of [3H]-adenosine at the low aff