Adenosine kinase from bovine adrenal medulla was purified 1600-fold by using ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration yielded a relative molecular mass around 42000 and Michaelis constants were 0.2 microM for adenosine and 20 microM for MgATP. The enzyme showed a broad specificity for purine nucleoside triphosphate as phosphate donors. Both free Mg2+ and ATP were inhibitors. AMP was a competitive inhibitor with regard to adenosine and a non-competitive inhibitor versus MgATP, while ADP was a uncompetitive inhibitor with regard to adenosine and a non-competitive inhibitor versus MgATP. Adenosine kinase was strongly inhibited by the bis(adenylyl) polyphosphates Ap4A and Ap5A. These compounds inhibited the enzyme competitively versus MgATP (Ki = 0.06 microM for Ap4A and 0.4 microM for Ap5A) and uncompetitively with regard to adenosine. The results of the kinetic analysis suggest an ordered bi-bi mechanism, adenosine being the first substrate. The phosphorylation of adenosine was unaffected in the presence of vanadate ions.