In a cytosolic extract from rat liver, the number and the concentration of ADP-binding sites as well as their dissociation constants were determined by using the rate-of-dialysis technique. Interfering cytosolic adenylate kinase was extracted from the cytosol by affinity chromatography on Ap5A-agarose, and remaining traces of enzyme activity were inhibited with (+)-catechin. Binding of ADP to cytosolic proteins was increased by poly(ethylene glycol) and decreased by EDTA. The effect of 0.1 mM-EDTA could be reversed by addition of equimolar concentrations of Mn2+ or Mg2+. In presence of 5% poly(ethylene glycol), added to increase local protein concentration, two binding sites for ADP were observed, with KD values of 1.9 microM (site I) and 10.8 microM (site II). The concentration of these binding sites, when extrapolated to cellular protein concentrations, were 30 microM (site I) and 114 microM (site II). It is concluded that a minimum of about 50% of total cytosolic ADP is bound to proteins, and that the ratio of free ATP/free ADP is at least twice that of total ATP/total ADP.