The microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) enzymes exist in a variety of cells and tissues, including liver, kidney, and testis. However, very little is known about brain epoxide hydrolases. Here we report the expression, localization, and subcellular distribution of mEH and sEH in cultured neonatal rat cortical astrocytes by immunocytochemistry, subcellular fractionation, Western blotting, and radiometric enzyme assays. Our results showed a diffuse immunofluorescence pattern for mEH, which colocalized with the astroglial cytoskeletal marker glial fibrillary acidic protein (GFAP). The GFAP‐positive cells also expressed sEH, which was localized mainly in the cytoplasm, especially in and around the nucleus. Western blot analyses revealed a distinct protein band with a molecular mass of ∼50 kDa, the signal intensity of which increased about 1.5‐fold in the microsomal fraction over the whole‐cell lysate and other subcellular fractions. The polyclonal anti‐human sEH rabbit serum recognized a protein band with a molecular mass similar to that of the affinity‐purified sEH protein (∼62 kDa), the signal intensity of which increased over 1.7‐fold in the 105,000g supernatant fraction over the cell lysate. Furthermore, the corresponding enzyme activities measured by using mEH‐ and sEH‐selective substrates generally corroborated the immunocytochemical and Western blotting data. These results suggest that rat brain cortical astrocytes differentially coexpress mEH and sEH enzymes. The differential subcellular localization of mEH and sEH may play a role in the cerebrovascular functions that are known to be affected by brain‐derived vasoactive epoxides. © 2008 Wiley‐Liss, Inc.