The characteristics for the binding of the selective cAMP phosphodiesterase inhibitor and antidepressant agent rolipram to brain and peripheral organs were investigated. (+/-)-[3H]Rolipram equilibrium binding and Scatchard analysis revealed saturable, reversible, stereospecific, Mg2+-dependent and heat-sensitive binding with an apparent Hill number of 1. Binding was detected both to membrane-bound and soluble sites, with dissociation constants Kd of 1.2 and 2.4 nM, respectively, and binding site concentrations (Bmax) of 19.3 and 23.6 pmol/g rat forebrain. The (-)-enantiomer of rolipram was ca. 20 times more effective than the (+)-enantiomer in displacing (+/-)-[3H]rolipram from membranes. Rolipram bound to brain tissue of all mammalian species tested including man, while tissue from bird and fish showed less binding. Organs other than brain exhibited only negligible binding. Only specific cAMP phosphodiesterase inhibitors (ICI 63.197, Ro 20-1724) were potent competitors, while rolipram itself was inactive in a variety of receptor binding assays of neuroactive ligands. The kinetics of (-)-[3H]rolipram binding to the particulate fraction revealed a complex association and dissociation behaviour. The nature of the rolipram binding protein(s) is not clear, but the low affinity binding site evident from binding kinetics may represent a rolipram-sensitive phosphodiesterase isoenzyme also common to some peripheral organs, while the high affinity binding site(s) may be related to PDE isoenzymes more confined to the central nervous system.