The functional significance of imidazoline I2 binding sites is unknown but microdialysis studies have indicated that the administration of I2-site ligands leads to an increase in extracellular levels of monoamines. The specific I2-site ligand 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) generates a cue in drug discrimination, thereby indicating functional consequences of I2-site ligand binding. In the present work, we explored the ability of selective noradrenergic and serotonergic ligands to substitute for 2-BFI. Hooded Lister rats were trained in two-lever operant chambers with condensed milk reward to distinguish 2-BFI (7 mg/kg) from saline vehicle, by pressing the correct lever to a predetermined success criterion. Training sessions were then interspersed with sessions in which animals were administered test substances and the proportion of lever presses on the 2-BFI-associated lever (substitution) recorded. Several agents exhibited significant partial substitution for 2-BFI: The monoamine-releasing agents D-amphetamine and fenfluramine dose-dependently substituted for 2-BFI, while norepinephrine (desipramine, reboxetine) and serotonin (clomipramine, citalopram) reuptake inhibitors substituted at one or more doses. Further investigation using specific receptor agonists and antagonists indicated a possible role for activation of alpha1-adrenoceptors but failed to support involvement of alpha2-adenoceptor, beta-adrenoceptor or 5-HT1A receptor activation. These results support the concept that the 2-BFI cue may contain both noradrenergic and serotonergic components.