We have determined the kinetics of dissociation of (R)-3-Quinuclidinyl (R)-4-[125I]Iodobenzilate ((R,R)-[125I]4IQNB) from muscarinic acetylcholine receptor preparations from the cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The dissociation curves are well described by a biexponential function and are consistent with subtype selectivity favoring slow dissociation from the M1, M3, and M4 receptors with a 20-fold faster dissociation rate for the M2 receptor. Following intravenous injection, (R,R)-[125I]4IQNB binds to receptor in the rat brain in concentrations which reflect the receptor concentration present in a structure. We determined the extent of radioligand present at two times, 2 and 24 hrs, as an indication of the relative proportions of m-AChR which exhibits rapid vs. slow dissociation of (R,R)-[125I]4IQNB. A good correlation between in vitro and in vivo results suggests that the relative populations of receptor subtypes can be imaged using in vivo pharmacokinetics of (R,R)-[125I]4IQNB.