Circadian rhythms have been described for numerous transmitter synthesizing enzymes in the brain but rarely in spinal cord. We measured spinal tyrosine-hydroxylase (TH) and nitric oxide synthase (NOS) levels in the thoracic intermediolateral nucleus, the location of sympathetic preganglionic neurons, in male wild type (WT) and dopamine D(3) receptor knockout mice (D(3)KO). TH and NOS levels both displayed circadian patterns in WT and D(3)KO animals with overall reduced TH and increased NOS expression in the D(3)KO mice. The circadian pattern of NOS expression was similar in WT and D(3)KO mice. In contrast, TH expression was inverted in D(3)KO mice, with TH levels consistently lower than in WT throughout the day, but strongly increased temporarily 1 h prior to daylight. TH is the rate-limiting enzyme for the production of dopamine. Spinal dopamine dysfunction is implicated in a sleep disorder called restless legs syndrome (RLS). RLS follows a circadian rhythm and is relieved clinically by dopamine D(3) receptor agonists. Our observations of an altered circadian pattern in spinal dopamine synthesis in D(3)KO animals may provide insight into putative dopaminergic mechanisms contributing to RLS.