The enzyme S-adenosyl-homocysteine hydrolase (AdoHcyase) which catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine is an adenosine binding protein. In the present study we examined the characteristics of [ 3 H]cAMP binding to purified AdoHcyase from bovine kidney in comparison with the high affinity adenosine binding site of AdoHcyase. AdoHcyase exhibits one [ 3 H]cAMP binding site with an affinity of K d =23.1+/-1.1nM and a B m a x of 116.6+/-3.8pmol/mg protein. Binding of [ 3 H]cAMP obeyed a monophasic reaction with a k + 1 value of 0.035min/M. The dissociation of AdoHcyase-[ 3 H]cAMP complex exhibited a time- and temperature-dependent character. After a 240min incubation at 0 o only 5-10%, however, at 20 o 90% were displaceable. Adenosine and cAMP displace each other with similar affinities of ec 5 0 57nM vs. ec 5 0 65nM. 2'-Deoxyadenosine, N 6 -methyladenosine, and NECA displace 25nM [ 3 H]cAMP and 10nM [ 3 H]adenosine with ec 5 0 values of 94, 90 and 80nM, respectively. All other nucleosides studied, adenine, inosine, adenosine-2',3'-dialdehyde, 2-chloroadenosine, aristeromycin, and adenine nucleotides were only week competitors for [ 3 H]cAMP and [ 3 H]adenosine. These compounds displace [ 3 H]cAMP and [ 3 H]adenosine with equal potencies. Our data indicate that the binding site for nanomolar concentrations of cAMP and adenosine at the AdoHcyase appears to be identical. The physiological implications of a cAMP binding site at the AdoHcyase remain to be established.