OBJECTIVE To establish recombinant human monoamine neurotransmitter including serotonin, norepinephrine, dopamine transporters (hSERT, hNET, hDAT) stably transfected cell lines and validate their binding and uptake function.METHODS Lipofectamine transfection was applied to transfect hSERT, hNET and hDAT into parental cell human embryo kidney (HEK)293.The transfected cells were stably selected by 800 ng· μl-1 G418 sulfate for about tow weeks, and the positive clones and their stabilities were identified by RT-PCR and Western blot methods.The binding and uptake function of hSERT, hNET and hDAT were identified by radioligand binding assay.RESULTS Results in RT-PCR and Western showed that HEK293-hSERT cell line was stably expressed hSERT RNA and protein in P1, P5, P10 and P15.Similar results were obtained in HEK293-hNET and HEK293-hDAT cells.Additionally, the hSERT expressed in HEK293-hSERT cell demonstrated significantly specific binding activity to SSRIs [3H]-citalopram.In the concenstration of 100 nmol·L-1 [3H]5-HT, the hSERT showed significantly 5-HT uptake function, and this action can be inhibited by 10 μmol· L-1 Fluoxetine.The parental cell HEK293 did not possess these functions.Similarly, HEK293-hNET and HEK293-hDAT showed specific binding activity to [3 H] nisoxetine (hNET) and [3 H] win35428 (hDAT), and uptake function to [3 H] NE (50 nmol· L-1) and [3 H]DA (50 nmol· L-1) which also can be inhibited by Desiparmine (10 μ mol· L-1) and Nomifensine (10 μmol· L-1).CONCLUSION The established HEK293-hSERT, HEK293-hNET and HEK293-hDAT cell lines which possess the function of endogenous hSERT, hNET and hDAT are stable functional monoamine neurotransmitter transporter cell lines.On account of the classic monoamine theory, these cell lines will be potent tools for screening and evaluation of antidepressant.