The regulation of adenosine phosphorylation by adenosine analogs was studied using highly purified human placental adenosine kinase [ATP: adenosine 5'-phosphotransferase (EC 2.7.1.20)]. Our observations lead us to classify the analogs into three groups as follows: type I, 5'-N-ethylcarboxamidoadenosine and 5'-methylthioadenosine; type II, N6-cyclohexyladenosine, N6-L-phenylisopropyladenosine, and 2-chloroadenosine; and type III, 6-methylmercaptopurine riboside. Type I compounds are inhibitors of adenosine kinase at 0.5 microM adenosine with IC50 values of 25 microM for 5'-N-ethylcarboxamidoadenosine and 250 microM for 5'-methylthioadenosine. These compounds stimulate adenosine kinase at 5.0 microM adenosine up to a maximum of 30 to 50% above basal velocity. They are not substrates for adenosine kinase. Type II compounds are inhibitors of adenosine kinase at 0.5 microM adenosine with an IC50 of 220 microM for N6-cyclohexyladenosine and 200 microM for N6-L-phenylisopropyladenosine. These analogs also stimulate adenosine kinase at 5.0 microM adenosine. 2-Chloroadenosine, N6-cyclohexyladenosine, and N6-L-phenylisopropyladenosine are phosphorylated by adenosine kinase with apparent Km values of 1,330, and 205 microM, respectively. 6-Methylmercaptopurine riboside (type III) inhibited enzyme activity with an IC50 of 10 microM at 0.5 microM adenosine and 215 microM at 5 microM adenosine and is a substrate for adenosine kinase. These data are consistent with the following: (a) 2-chloroadenosine, N6-cyclohexyladenosine, and N6-L-phenylisopropyladenosine may not be good adenosine receptor agonists in vivo because they are phosphorylated into active derivatives by adenosine kinase; (b) 5'-N-ethylcarboxamidoadenosine and 5'-methylthioadenosine are superior candidates for adenosine receptor agonists in vivo because they are not phosphorylated; (c) 5'-N-ethylcarboxamidoadenosine, 5'-cyclohexyladenosine, N6-L-phenylisopropyladenosine, and 2-chloroadenosine may interact with adenosine kinase at two sites on the enzyme, a catalytic site and a regulatory site; and (d) 6-methylmercaptopurine riboside may interact with the enzyme at the catalytic site only.