The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23 degrees of 3.75 X 10(7) M-1 min-1 and a first-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd for [3H]PDBU binding varied only slightly with temperature. From its temperature dependence, [3H]PDBU binding appeared to be associated with a small increase in enthalpy (delta H degrees = +0.4 kcal/mol) and a large increase in entropy (delta S degrees = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30 degrees to 62 min at 4 degrees. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethyleneglycolbis(beta-aminoethyl ether)N,N'-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.