Oestrogen receptors (ERs) are important for sexual differentiation of the brain. Previous studies in rats have reported that the locus coeruleus (LC), a catecholaminergic nucleus in the brain stem, is sexually dimorphic such that females have more neurones than males. We hypothesised that ERs may be important for sexual differentiation of this nucleus in mice. Because previous studies reported conflicting results regarding ER protein expression in the mouse LC, we evaluated ER alpha and ER beta gene expression by in situ hybridisation and the real-time reverse transcription-polymerase chain reaction. We demonstrated that both ER alpha and ER beta mRNAs are present in tyrosine hydroxylase-immunoreactive (TH-ir) cells in the male LC. In the female LC, ER alpha mRNA is present at levels similar to males, whereas ER beta mRNA expression is significantly lower than in males. Similar to rats, male mice have fewer TH-ir cells in the LC than females at 60 days after birth, but the difference is absent at 120 days after birth when females exhibit a similar reduction in TH-ir cells. The transient sex difference is ER beta-dependent because is it absent in ER beta knockout mice, and is due to regulation of TH expression and not from death of TH-ir cells. Testicular hormones produced at adolescence are necessary for the regulation of TH expression in the male LC because orchidectomy of pre-pubertal males prevented the decrease in TH-ir cells, whereas treatment of gonadectomized males with testosterone or its metabolite, 5 alpha-androstan-3beta,17beta-diol, restored the intact male phenotype. Overall, these studies indicate that ER beta is important in regulating TH expression in the mouse LC.