It has been proposed that the extrapyramidal symptoms such as tardive dyskinesia developed by patients on long-term haloperidol treatment may be the result of uptake of haloperidol metabolites into neurons via the monoamine neurotransmitter transporters followed by neurotoxic events, as occurs for MPP+, the pyridinium metabolite of MPTP. We recently showed that haloperidol and its metabolites are inhibitors of the human noradrenaline transporter (NAT), dopamine transporter (DAT) and serotonin transporter (SERT), and determined their K i values for inhibition of the three transporters expressed in transfected COS-7 cells. In this study, we extended the investigation of these compounds to their inhibitory effects on DAT, SERT and the high affinity choline uptake (HACU) in neuronal cultures from embryonic rat brain, and investigated whether the compounds are substrates or non-transported inhibitors of the NAT, DAT and SERT in transfected COS-7 cells and DAT and SERT in the neuronal cultures. Haloperidol and its metabolites inhibited DAT, SERT and HACU in the neuronal cultures, indicating that they are not specific inhibitors of the monoamine neurotransmitter transporters. The ratio of the K i values of the least and most potent inhibitors were found to be 2.8 for DAT, 24 for SERT and 7.6 for HACU. The compounds were more potent inhibitors of DAT and SERT in neuronal cultures than we found previously in transfected COS-7 cells. The question of whether the compounds are substrates or non-transported inhibitors of the monoamine transporters was investigated by determining whether they caused an increase in efflux of [3H]amine in transfected COS-7 cells or neuronal cultures preloaded with [3H]amine. Haloperidol metabolites were weak substrates for SERT, but not for NAT or DAT, in transporter-transfected COS-7 cells. In neuronal cultures, the metabolites appeared to be non-transported inhibitors or very weak substrates of DAT and SERT. Despite inhibition of the monoamine transporters by haloperidol and its metabolites, there is little evidence to support the proposal that these compounds are likely to cause neurotoxic effects via neuronal uptake using the monoamine transporters. The mechanisms of the side effects of haloperidol therapy, such as tardive dyskinesia, are still unclear, but are unlikely to depend on interactions of the drug or its metabolites with NAT, DAT or SERT.