Adenosine inhibits the rat liver adenylate cyclase system at a regulatory site that is distinct from the glucagon receptor, the guanine nucleotide regulatory site, and the active site involved in catalysis of ATP to cyclic AMP. The effects of the nucleoside are also independent of the concentration of uncomplexed ATP (ATP4-) in the assay medium. Glucagon, but not guanine nucleotides, sensitizes the system to inhibition by adenosine. Depending on assay conditions, the hormone can shift the concentration of adenosine required for 50% inhibition by as much as 10-fold. Under optimal conditions, the apparent Ki for adenosine is 25 micron. Both Mg2+ and Mn2+ increase adenylate cyclase activity and, in order of relative potency, increase the sensitivity of the enzyme to adenosine inhibition; Mn2+ is 50- to 100-fold more potent than Mg2+. The adenosine inhibitory site exhibits stringent structural requirements for nucleoside action. Most alterations of the purine ring result in loss of activity, whereas alterations in the ribose ring are tolerated, and some deoxyadenosine analogs are even more effective than adenosine. Naturally occurring nucleosides and nucleotides, such as inosine, guanosine, and 5'-AMP, are inactive. Analog studies reveal also that inhibition of the hepatic system occurs at a site which is clearly different from the sites through which adenosine activates other adenylate cyclase systems, and that the liver enzyme appears to have no site for activation by the nucleoside.