Cholesterol is required in the brain for synaptogenesis and its turnover is critical for cerebral functions. Several proteins involved in cholesterol handling and metabolism are transcriptionally regulated by the nuclear liver X receptor (LXR) alpha and beta. Sterol 27-hydroxylase (CYP27) is a ubiquitously expressed enzyme involved in cholesterol metabolism. Notably, its deficiency causes a disease characterized by progressive neurologic impairment. With the final goal to understand the pathophysiological role of CYP27A1 in the CNS, we studied the expression pattern of Cyp27a1 and other related genes in primary cultures of rat glia and neurons. Secondly, given the pivotal role of LXR in the regulation of cholesterol homeostasis, we investigated the effects of its activation on the expression of Cyp27a1.We found that primary astrocytes express different sterol hydroxylases and are able to uptake exogenous 27-hydroxycholesterol. We found that both microglia and astrocytes express preferentially Lxrbeta. However, despite this similarity, we observed cell-specific responsiveness of known and novel (including Cyp27a1) target genes to LXR activation. The increase of mRNA and protein levels in treated astrocytes is paralleled by transactivation of the proximal Cyp27a1 promoter in transfected astrocytes. We suggest that the astrocyte-restricted up-regulation of Cyp27a1 may be ascribable to differential expression of transcriptional co-activators. Given the role of astrocytes in maintaining brain homeostasis, we hypothesize that impairment of CYP27 activity in these cells may alter critical features of the astrocytes, from the handling and delivery of cholesterol to neurons to the release of signaling molecules.