The metabolism of 27-, 25- and 24-hydroxycholesterol in cultures of rat astrocytes, Schwann cells and neurons was studied. 27- and 25-Hydroxycholesterol, but not 24-hydroxycholesterol, underwent 7α-hydroxylation with subsequent oxidation to 7α-hydroxy-3-oxo-Δ4 steroids in all three cell types. When cells were incubated for 24 h with 0.28 nmol of 27-hydroxycholesterol in 10 ml of medium, the rates of conversion into 7α-hydroxylated metabolites were 0.21, 0.12 and 0.02 nmol/24 h per 106 cells in the media of astrocytes, Schwann cells and neurons respectively. The corresponding values for 25-hydroxycholesterol were 0.26, 0.16 and 0.04. A minor fraction of 27-hydroxycholesterol and its 7α-hydroxylated metabolites was oxidized to 3β-hydroxy-5-cholestenoic acid, 3β,7α-dihydroxy-5-cholestenoic acid and 7α-hydroxy-3-oxo-4-cholestenoic acid. In addition to the two hydroxycholesterols, other 3β-hydroxy-Δ5 steroids, dehydroepiandrosterone, pregnenolone, 3β-hydroxy-5-cholestenoic acid and 3β-hydroxy-5-cholenoic acid underwent 7α-hydroxylation. Competitive experiments did not distinguish between the presence of one or several 7α-hydroxylases. In astrocyte incubations, 27-hydroxycholesterol also underwent 25-hydroxylation, and 12% of its metabolites carried a 25-hydroxy group. 25-Hydroxylation of added 24-hydroxycholesterol was also observed in the astrocyte incubations, as was the formation of 7α,25-dihydroxy-4-cholesten-3-one, 25-hydroxycholesterol and 7α,25-dihydroxycholesterol from endogenous precursor(s). Our study indicates that side-chain oxygenated cholesterol can undergo metabolic transformations that may be of importance for cholesterol homoeostasis in the brain.