Evidence indicates that monoaminergic neurotransmitter transporters are promiscuous, transporting substrates other than their cognate neurotransmitters. For example, serotonin is transported by the dopamine transporter (DAT) under conditions in which serotonin transporter (SERT) activity is eliminated (e.g., pharmacological inhibition). We performed a kinetic analysis of [(3)H]serotonin uptake in rat striatal synaptosomes (expressing DAT and SERT) and hippocampal synaptosomes (expressing SERT, but not DAT). Nonspecific [(3)H]serotonin uptake was defined as the amount of uptake remaining in the presence of fluoxetine (10microM) or paroxetine (0.05microM). In hippocampal synaptosomes, K(m) and V(max) values for [(3)H]serotonin uptake did not differ whether fluoxetine or paroxetine was used to define nonspecific uptake. However, in striatal synaptosomes, both K(m) and V(max) values for [(3)H]serotonin uptake were greater when fluoxetine, rather than paroxetine, was used to define nonspecific uptake. These data suggest that, at the concentrations employed, fluoxetine inhibits serotonin uptake at both DAT and SERT, whereas paroxetine only inhibits serotonin uptake at SERT. Thus, when DAT is inhibited by GBR 12909, kinetic parameters for serotonin uptake via SERT in striatum are not different from those obtained in hippocampus. These findings have important implications regarding the analysis of monoaminergic reuptake in brain regions exhibiting heterogeneous transporter expression.