The potential for noninvasive quantification of myocardial muscarinic receptors using PET data, a mathematical model, multi-injection protocols and 11C-labeled methylquinuclidinyl benzilate (MQNB) as a radioligand was previously demonstrated in dogs. The present study examines the possibility of optimizing the experimental protocol to make this approach suitable for human studies. For six normal subjects, the protocol included three injections: a tracer injection, followed 30 min later by an injection of an excess of unlabeled MQNB (displacement) and then 30 min later by a simultaneous injection of unlabeled and labeled MQNB (coinjection). The model input function was estimated from the PET data corresponding to the left ventricular cavity. This protocol enables a separate evaluation of all parameters of a ligand-receptor model which includes three compartments and seven parameters. The complexity of this three-injection protocol, however, appears to be inconvenient for clinical use. A simplified two-injection protocol (tracer injection and coinjection) was evaluated in five other normal subjects and the results were compared to those obtained with the three-injection protocol. In regions of interest over the left ventricle, the mean value of the receptor concentration B'max and the equilibrium dissociation constant Kd were 26 +/- 7 pmole/ml tissue and 2.0 +/- 0.5 pmole/ml tissue, respectively. The possible existence of nonspecific binding was studied in two subjects using a double-displacement protocol. The corresponding rate constant was found to be very low (0.03 min-1).