An approximate 140-fold purification of the A 1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A 1 adenosine receptor antagonist. As assessed by [ 3h]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol mg protein. Interaction of solubilized A 1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [ 3h]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 ± 22 pmol mg protein with typically 5–15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A 1 adenosine receptor: R-PIA > S-PIA >62; NECA. In purified preparations, the photoaffinity probe [ 1251]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38000 which has previously been shown to be the A 1 adenosine receptor binding subunit.