Quantitative autoradiography was used to characterize and localize [3H]cGMP binding sites in the rat brain. [3H]cGMP binding was found to be pH-sensitive (with two optima at 7.4 and 5.0) and Mg2+-dependent. At pH 7.4, the binding was dependent on inclusion of the phosphodiesterase inhibitor IBMX. In contrast, at pH 5.0, IBMX had little effect on binding. The binding of [3H]cGMP was reversible and saturable with a Kd of 22 nM at pH 7.4 and 36 nM at pH 5.0. Bmax values were 172 fmol/mg at pH 7.4 and 462 fmol/mg at pH 5.0. [3H]cGMP binding was inhibited by cGMP and its analogues, with cGMP and cAMP being the most potent at pH 7.4 and cGMP and 8-Br-cGMP being the most potent at pH 5.0. Using an extracellular pH 7.4 buffer, the selective cGMP-dependent protein kinase (PKG) inhibitor Rp-8pCPT-cGMPS had very little effect on [3H]cGMP binding. In contrast, with a cytosolic pH 5.0 buffer, Rp-8pCPT-cGMPS displaced binding in the cerebellum. This indicates that PKG is localized in the cerebellum, and that the binding to PKG is favored under cytosolic conditions. Autoradiographic localization of [3H]cGMP binding sites revealed a heterogeneous distribution with the highest densities in the substantia nigra and interpeduncular nucleus. High densities were also observed in the basal ganglia, the medial habenular nucleus, the frontoparietal cortex, the lateral amygdaloid nucleus and the subiculum. It is concluded that the nature of [3H]cGMP binding is complex, with one site probably being related to cytosolic PKG mainly found in the cerebellum, and one site probably representing cGMP-stimulated phosphodiesterase mainly located in the forebrain.