Oestrogen modulates cognitive function and affective behaviours subserved by the prefrontal cortex (PFC). Identifying and localising oestrogen receptor (ER)alpha, in human PFC will contribute to our understanding of the molecular mechanism of oestrogen action in this region. Inferences about the site of action of oestrogen in human brain are derived largely from studies performed in nonhuman mammalian species; however, the congruence of findings across species has not been demonstrated. Furthermore, the laminar, cellular, and subcellular localisation of ERalpha in the cortex is debated. Therefore, we compared the distribution of ERalpha in human dorsolateral prefrontal cortex (DLPFC) with that of monkey DLPFC and rat medial PFC. Immunohistochemistry performed on frontal cortex from the three species demonstrated ERalpha positive cells throughout all layers of the PFC, in pyramidal and nonpyramidal neurones, with both nuclear and cytoplasmic immunoreactivity. Western blot analyses and preabsorption studies confirmed that the antibody used recognised ERalpha and not ERbeta. A strong ERalpha immunoreactive band corresponding to the full-length ERalpha protein (65-67 kDa) in the frontal cortex of all three species matched the size of the predominant immunoreactive band detected in breast cancer cell lines known to express ERalpha. Additionally, other ERalpha immunoreactive proteins of varying molecular weight in breast cancer cells, rat ovary and mammalian brain were detected, suggesting that ERalpha may exist in more than one form in the mammalian frontal cortex. The present study provides evidence that ERalpha protein exists in neurones in mammalian PFC and that ERalpha is anatomically well-positioned to directly mediate oestrogen action in these neurones.