Riboflavin inhibited binding of both agonist and antagonist radioligands to rat brain A 1adenosine receptors with K i values of approximately 10 Β΅M. In an adenylate cyclase assay with membrane preparations from either rat adipocytes or DDT MF-2 cells, both of which contain A 1 -adenosine receptors, riboflavin inhibited isoproterenol-stimulated cyclase activity with an IC 50 of approximately 20 Β΅M. However, the inhibition of cyclase by riboflavin was not reversed by an A 1 -selective antagonist, nor by pretreatment with pertussis toxin. Thus, neither A 1 -receptors nor G i -proteins appear critically involved in the inhibition of cyclase by riboflavin. Riboflavin did block the stimulation by an adenosine analog of [ 35 S]GTPΞ³S binding in rat cerebral cortical membranes. However, riboflavin also inhibited the stimulation by fMLP of [ 35 S]GTPΞ³S binding in HL-60 cell membranes. Riboflavin inhibited forskolin-stimulated cyclase in membranes from DDT MF-2 cells > rat adipocytes > PC12 cells, hamster CHO M2 cells, and wild-type S49 cells. There was virtually no inhibition of forskolin-stimulated cyclase in membranes of human platelets, rat cerebral cortex, or cyc -S49 cells lacking G s -proteins. The calcium-stimulated cyclase in rat cerebral cortical membranes was inhibited by riboflavin. A preincubation of membranes with riboflavin markedly enhanced the inhibition for DDT MF-2 and wild-type and cyc -S49 membranes. The extent of inhibition in the different cell lines was dependent on the agent used to stimulate cyclase. Riboflavin, like the P-site inhibitor 2Β΄,5Β΄-dideoxyadenosine, was more potent and efficacious when manganese instead of forskolin was used as the stimulant. However, unlike the P-site inhibitor, riboflavin did not markedly inhibit GppNHp-or fluoride-stimulated cyclase. Riboflavin at low micromolar concentrations appears to have three possibly interrelated effects on second messenger systems subserved by G-proteins. These are antagonism at A 1 -adenosine receptors, inhibition of turnover of guanyl nucleotides at G-proteins, and inhibition of adenylate cyclase. Drug Dev. Res. 42:98-108, 1997.