The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.