Dehydroepiandrosterone (DHEA) is an important neurosteroid with multiple functions in the central nervous system including neuroprotection. How DHEA exerts its neuroprotection function has not been fully elucidated. One possible mechanism is via its active metabolites, 7alpha-OH DHEA and 7beta-OH DHEA. The purpose of this research is to understand how DHEA is metabolized to 7alpha-OH DHEA and 7beta-OH DHEA by brain tissue. DHEA was incubated with rat brain microsomes and mitochondria and the 7alpha-OH DHEA and 7beta-OH DHEA formed by these fractions were analyzed by LC/MS. For the first time, we observed that DHEA could be metabolized to 7alpha-OH DHEA and 7beta-OH DHEA in mitochondria but the formation of 7alpha-OH DHEA and 7beta-OH DHEA demonstrated different enzymatic kinetic properties. Adding NADPH, an essential cofactor, to mitochondria incubation mixtures increased only the formation of 7alpha-OH DHEA, but not that of 7beta-OH DHEA. Addition of estradiol to the incubation mixtures inhibited only the formation of 7alpha-OH DHEA, but not that of 7beta-OH DHEA. Western blot analysis showed that both microsomes and mitochondria contained cytochrome P450 7B. We also found that 7alpha-OH DHEA could be converted to 7beta-OH DHEA by rat brain homogenates. Our data suggest that 7alpha-OH DHEA and 7beta-OH DHEA are formed by different enzymes and that 7beta-OH DHEA can be formed from both DHEA and 7alpha-OH DHEA, although the overall level of 7beta-OH DHEA was very low.