Neuroendocrine-specific protein (NSP) reticulons (RTN) are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. At least 4 different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are still elusive. In the present study, the expression of human reticulon 3 (hRTN3) in a variety of tissues was investigated. Northern blotting hybridization revealed higher expression of hRTN3 in normal brain and some endocrine-related organs such as thyroid relative to other non-endocrine organs. Twelve surgical specimens meeting the histological criteria for astrocytoma grade II, III and IV were investigated by in situ hybridization and immunohistochemical analysis, which demonstrated the overexpression of hRTN3 in astrocytoma tumor cells, while in non-cancerous brain tissues hRTN3 was mainly expressed in neurons and almost no signals in glia cells could be detected. The differential expression of hRTN3 between astrocytoma and non-cancerous tissue may provide insight into the progress of astrocytoma.