The aim of this study is to investigate the pathway of diencephalic dopaminergic (DA) neuronal innervating into the spinal cord in mice, the pathway is postulated relevant to clinical restless legs syndrome (RLS). Tyrosine hydroxylase (TH) immunohistochemistry was used to identify the DA neuron. The fluorescent tracer Fluoro-Gold (FG) was stereotaxically injected into the T10-L5 spinal cord of CBL57 mice (n=20) seven days before the animals were sacrificed. The diencephalic sections were stained with TH antibody and the FG tracer present in the diencephalic DA neurons were examined under fluoresce microscope. The average number of total DA neurons per side in A11, A12, A13 and A14 was 66+/-8, 221+/-12, 350+/-17 and 254+/-21 respectively. After being injected into the spinal cord, FG reached the DA neurons within the A10 and A11 groups, but didn't target to any other DA neuron groups including the A8 and A9 groups in substantia nigra (SN). The diencephalic A11 DA neurons possessed long axons extending over several segments and possibly traversing the entire length of the spinal cord. It is the first time to report A10 and A11 DA neuron projections into the spinal cord in mice.