The fraction of transporters occupied following injection of specific inhibitors is an important parameter for defining and comparing the molecular mechanisms of different drugs. This work generalizes the reference tissue method to estimate dopamine transporter occupancy for two levels of cocaine administration using only a single injection of [18F]FECNT. The results are validated by comparison with literature values. Five rhesus monkeys were studied. On each animal, a baseline scan was collected following [18F]FECNT injection (phase a). At 120 min postinjection, 0.1 mg/kg cocaine was injected and the animal was scanned for 50 additional min (phase b). Then 1.0 mg/kg cocaine was injected and another 50-min scan sequence was collected (phase c). Time–activity curves (encompassing all three phases) were generated for each animal from regions drawn over the putamen and cerebellum. The putamen curve was modeled using the cerebellum as the input function. Percent DAT occupancy following the cocaine injections was determined by comparing k3/k4 = Bmax/kD for the three phases. The 0.1 and 1.0 mg/kg cocaine doses occupied 53% ± 5% and 87% ± 5% of the transporters, respectively. The measured occupancies are consistent with literature values that maintain self-administration in animals and produce a “high” in human subjects. This work demonstrates that a single injection of [18F]FECNT can be used to measure the effects of multiple cocaine challenges. Two advantages of this technique are: reduced variability in dose–response curves because the subject is his/her own control, and the 18F label allows evaluation of longer-acting drugs. Synapse 44:203–210, 2002. © 2002 Wiley-Liss, Inc.