The affinity of adenosine for the human adenosine A1 receptor expressed on Chinese hamster ovary cell membranes has been measured in the presence and absence of GTP. The competitive effect of endogenous adenosine on the binding properties of adenosine A1 receptors was estimated from differences in the binding of N6-cyclohexyladenosine measured in the absence and presence of adenosine deaminase. From these data, the affinity of adenosine for the high- and low-affinity states of the human adenosine A1 receptor (7 x 10(7) and 1.3 x 10(5) M-1, respectively) was calculated.