Investigations on the effect of normal healthy ageing on the muscarinic system have shown conflicting results. Also, in vivo determination of muscarinic receptor binding has been hampered by a lack of subtype selective ligands and differences in methods used for quantification of receptor densities. Recent in vitro and in vivo work with the muscarinic antagonist (R,R)-I-QNB indicates this ligand has selectivity for m(1) and m(4) muscarinic receptor subtypes. Therefore, we used (R,R)[(123)I]-I-QNB and single photon emission tomography to study brain m(1) and m(4) muscarinic receptors in 25 healthy female subjects (11 younger subjects, age range 26-32 years and 14 older subjects, age range 57-82 years). Our aims were to ascertain the viability of tracer administration and imaging within the same day, and to evaluate whether normalization to whole brain, compared to normalization to cerebellum, could alter the clinical interpretation of results. Images were analyzed using the simplified reference tissue model and by two ratio methods: normalization to whole brain and normalization to cerebellum. Significant correlations were observed between kinetic analysis and normalization to cerebellum, but not to whole brain. Both the kinetic analysis and normalization to cerebellum showed age-related reductions in muscarinic binding in frontal, orbitofrontal, and parietal regions. Normalization to whole brain, however, failed to detect age-related changes in any region. Here we show that, for this radiotracer, normalizing to a region of negligible specific binding (cerebellum) significantly improves sensitivity when compared to global normalization.