Antisera against the non-selective adenosine receptor agonist 5'-N-ethylcarboxamido-adenosine (NECA) have been raised by immunizing rabbits with NECA-coupled bovine serum albumin. The antisera which bind [3H]NECA with high affinity were purified by affinity chromatography using NECA-coupled Sepharose as the affinity gel without significant changes in [3H]NECA-binding properties. The order of the affinity for various adenosine receptor ligands of the purified or unpurified antisera was 5'-N-cyclopropylcarboxamidoadenosine > or = NECA > 2',5'-dideoxyadenosine > 2-chloroadenosine > theophylline > isobutylmethylxanthine > (R)-N6-phenylisopropyladenosine = N6-cyclohexyl- adenosine. This specificity was found to be similar to that of the non-receptor NECA-binding sites, which had been found in various tissues such as human placentas and mouse P815 mastocytoma cells, rather than to that of adenosine receptors. These anti-NECA antibodies will be useful as immunochemical agents in the search for endogenous ligand(s) to the non-receptor NECA-binding sites.