Administration of phenserine, an acetylcholinesterase inhibitor, raises endogenous brain acetylcholine levels and has been previously shown to reduce in vivo binding of the muscarinic cholinergic receptor antagonist [(11)C]N-methylpyrrolidinyl benzilate ([(11)C]NMPYB) in the awake rat brain. In this study, phenserine pretreatment was studied in both awake and isoflurane-anesthetized rats using the techniques of ex vivo dissection or in vivo microPET imaging. In ex vivo dissection experiments, a statistically significant 10% inhibition of [(11)C]NMPYB binding could be demonstrated in both awake and anesthetized animals after phenserine pretreatment, showing no deleterious effect of using isoflurane anesthesia. However, microPET imaging in anesthetized animals failed to successfully demonstrate inhibition of [(11)C]NMPYB binding following the identical phenserine treatment protocol. These results demonstrate that in small numbers of subjects ex vivo dissection may be a more sensitive experimental method for determining small changes of in vivo radiotracer binding in this model of neurotransmitter competition for brain receptor sites.