[3H]Spiroperidol (spiperone) binding in the presence of mianserin, a serotonin (5-HT2) receptor antagonist, was characterized in rat brain using quantitative autoradiography. All binding parameters were directly determined from film densities. Competition and kinetic studies revealed that [3H]spiroperidol binds to a site having characteristics of the dopamine, D2, receptor in striatum. The general binding parameters were similar to values obtained in homogenate and swabbed section studies except as related to agonist binding and guanine nucleotide sensitivity. Competition studies with dopamine revealed biphasic competition curves with a Kh of 8.23 nM and a Kl of 12.3 microM. The percentage of high-affinity sites was 90%. Guanine nucleotides (1 microM guanylyl-imidodiphosphate) completely converted the high-affinity site to a low-affinity site. Quantitative regional distribution studies revealed high binding in striatum, olfactory tubercle and nucleus accumbens, with lower binding in other dopamine innervated regions including frontal and cingulate cortex. [3H]Spiroperidol was also found to bind to a spirodecanone site with an anatomical localization distinct from the dopamine and serotonin systems and in a region (entorhinal cortex) not previously reported. This report provides a detailed pharmacologic and regional characterization of [3H]spiroperidol binding to D2 receptor in rat brain using quantitative autoradiography to determine all binding parameters. This report also demonstrates an increased percentage of sites in the high-affinity state of the D2 receptor in tissue sections and increased affinity of the guanine regulatory protein for guanine nucleotides.