In addition to the neurosteroids pregnenolone and dehydroepiandrosterone, organic solvent extracts of rat brains contain related compounds that react with various reagents to yield additional amounts of these ketosterolds. Among the chemicals producing these increments are triethylamine, HCI, FeCl3, and Pb(OAc) In a recent paper (1), estimates of the concentrations of five steroids and their conjugates in mammalian brains were recorded. Also described were the techniques used for the separation, identification, and quantification of these socalled neurosteroids. An unusual feature of the reported findings was the relatively high concentrations of steroidal conjugates, which were designated "sulfolipids." Following from our previous experience with analogous conjugates of cholesterol (2), we found that there appear to exist in mammalian brain nonpolar conjugates of neurosteroids, particularly pregnenolone (3f3hydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3f-hydroxy-5-androsten-17-one).These, when treated with an organic base such as triethylamine or pyridine, were converted to a steroid sulfate that could be identified and quantified (after acid hydrolysis) as the free steroid. Some of these conjugates appeared to be present in relatively high concentrations in brain, suggesting that these conjugates play a physiological role in the nervous system.The experiments described in this paper bear on these substances. More The second part was separated into its ketonic and nonketonic (part B) components by treatment with polystyrenebenzyloxime resin to remove carbonyl compounds (8), including the endogenous ketosteroids and their derivatives. Sample B was dissolved in 20 ml of benzene containing 2 ml of acetic acid and a bolus of 200,000 dpm of tritiated pregnenolone. The oxime resin (100 mg) was added, and the suspension was stirred for 2 hr at room temperature. A check of the tritium content of the supernatant revealed that >90% of the tritiated steroid had bound to the resin. The reaction was allowed to proceed to Abbreviation: P450scc, side chain-cleaving P450.