Neurosteroids synthesized within the central nervous system play essential roles in modulating neurotransmission, providing neuroprotection, regulating immune responses, influencing behavior and cognition and mediating stress physiology. Despite their broad significance, the specific brain cell types capable of de novo steroid synthesis from cholesterol remain poorly defined. In this study, we analyzed single-cell transcriptomic data to map steroidogenic gene expression across cell populations in the murine brain, focusing on the de novo production of the neurosteroid pregnenolone. Our findings reveal that de novo steroidogenesis, as marked by Cyp11a1 expression, is predominantly confined to specific neuronal subtypes, particularly glutamatergic neurons of the intra- and extra-telencephalic regions and the corticothalamic layer. In contrast, Star expression, which is essential for mitochondrial cholesterol import, was more broadly distributed, occurring in both neuronal and non-neurona