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This study aimed to describe the expression of oestrogen receptor (ER)α, ERβ and aromatase in salivary gland adenoid cystic carcinoma (ACC).ERα, ERβ and aromatase expression was analysed by immunohistochemistry in tissue microarray blocks from 38 cases of ACC and seven normal salivary glands. The intracellular localization and amount of total protein expression were investigated by immunofluorescence and western blotting in an ACC cell line. Western blotting analysis showed overexpression of ERα, ERβ and aromatase in the ACC cell line; however, with immunofluorescence, only ERβ was shown to be expressed in the nucleus. Immunohistochemistry revealed positive nuclear expression of ERβ, positive cytoplasmic expression of aromatase and a lack of ERα expression as compared with normal salivary glands.The nuclear expression of ERβ indicates that oestrogen may be active in ACC and possibly able to mediate E2-targeted gene transcription. This study strongly suggests that ERβ may be involved in tumour progression, playing a role in tumour development, and thus corroborating the indication for ER antagonists in the clinical control of ACC. This study opens a new perspective on the potential use of anti-oestrogens and aromatase inhibitors as therapeutic agents against ACC.