Signal transduction by A1 adenosine receptors was investigated by measuring the modulation by adenosine agonists of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP[S]) binding to guanine nucleotide-binding proteins (G proteins). The extent of stimulation of [35S]GTP[S] binding was dependent on the presence of high concentrations of Mg2+ (1-10 mM), GDP (10 microM), and NaCl (100 mM). Under optimal conditions, the agonist (R)-N6-phenylisopropyladenosine [(R)-PIA] stimulated binding of [35S]GTP[S] to G proteins approximately 2.3-fold. All adenosine receptor agonists tested stimulated the binding of [35S]GTP[S] with a rank order of potency typical of A1 adenosine receptors in bovine tissues, (R)-PIA > 2-chloro-N6-cyclopentyladenosine > (S)-PIA > 5'-N-ethylcarboxamidoadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e. The EC50 values for G protein activation correlated with the Ki value of agonists for inhibition of radioligand binding to the high affinity state of the A1 adenosine receptor. The inclusion of 100 mM NaCl as well as increasing GDP concentrations led to a parallel increase of Ki values and EC50 values. In addition, both compounds induced a shift of A1 adenosine receptors from the high affinity state for agonists to the low affinity state.