18F-labeled (-)fluoroethoxybenzovesamicol [(-)FEOBV] is a novel PET tracer which binds to the vesicular acetylcholine transporter of cholinergic neurons. To evaluate the in vivo binding specificity and kinetic properties of (-)FEOBV, studies were performed in isolated working rat hearts. External gamma,gamma-coincidence monitoring of hearts indicated high extraction of radiotracer by the myocardium and rapid wash-out of unbound tracer (> 90% of maximal accumulation) within 5 min. Inclusion of (-)vesamicol (10 microM) throughout perfusion decreased the retention of (-)FEOBV by 71% (P < 0.005) and 76% (P < 0.005) in atria and ventricles, respectively. However, the initial uptake rate of the tracer was unaffected. Additional experiments showed the inactive stereoisomer, (+)FEOBV, to have a lower retention than the (-)FEOBV isomer in ventricles, indicating stereospecificity of the binding process that is consistent with structure-activity relationships of vesamicol congeners. The results indicate (-)FEOBV to be a moderately specific probe of vesamicol-sensitive binding in cholinergic neurons of the heart in experimental conditions that assure adequate washout of unbound tracer. However, the utility of the radiotracer for in vivo studies with PET is likely to be limited by the low rate of specific binding in myocardium consistent with the low density of cholinergic neurons in the heart.