Steroids in the brain arise both from local synthesis and from peripheral sources, and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. Following extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3,5-tetrahydrodeoxycorticosterone, testosterone and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3,5-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone and 5-dihydroprogesterone. Further, the presence of 3-, 11-, 17- and 20-hydroxylated metabolites of 3,5-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20-reduced metabolites could also be found for pregnenolone, progesterone and 5-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3,5-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.