Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: Km = 3 X 10(-7) M for NE, Km = 9 X 10(-8) M for DA, and Km = 3 X 10(-8) M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with Ki = 2.5 X 10(-6) M for NE uptake, Ki = 2.0 X 10(-6) M for DA uptake, and Ki = 1 X 10(-5) M for 5-HT uptake. These constants compare well IC50 values for the same transmitters: NE, IC50 = 2.4 X 10(-6) M; DA, IC50 = 2.8 X 10(-6) M; 5-HT, IC50 = 1.0 X 10(-5) M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.