Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABAA receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5α-reductase (5α-R) type I and 3α-hydroxysteroid dehydrogenase (3α-HSD). This study evaluates 5α-R type I and 3α-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100β immunohistochemistry. We demonstrate that 5α-R type I and 3α-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5α-R type I nor 3α-HSD mRNAs are expressed in S100β- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5α-R type I and 3α-HSD in cortical and hippocampal GABAergic interneurons. However, 5α-R type I and 3α-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABAA receptors, either with an autocrine or a paracrine mechanism or by reaching GABAA receptor intracellular sites through lateral membrane diffusion.