We have previously characterized specific binding of the phorbol ester tumor promoter [20-3H]phorbol 12,13-dibutyrate to several tissues, including mouse skin and brain. We report here that specific binding activity in chicken brain increases dramatically during development. In whole embryonic chicken brain, binding increased from 2.0 to 6.8 to 10.6 pmol/mg of protein at 7, 14, and 20 days, respectively. Adult chicken brain bound 15 pmol/mg of protein. In contrast to specific binding activity, binding affinity remained constant. Substantial regional localization of binding activity within the brain was found. For calf brain, representative values ranged from 4.6 pmol/mg for medulla to 38.1 pmol/mg for frontal lobe. Binding affinity did not vary. Inhibition of binding by postulated neurotransmitters and antagonists was examined. D-Propranolol and quinidine, membrane-stabilizing drugs, inhibited binding competitively, although at mM concentrations.