Pregnenolone (PRG), a major neurosteroid, suppressed carbachol-induced salivary secretion in perfused submandibular gland in rats. These effects were enhanced and depressed by agonistic muscimol (MUS) and antagonistic bicuculline to the γ-aminobutyric acid A receptor (GABA_A-R), respectively. In contrast, PRG-sulfate, a sulfate-conjugated PRG metabolite, antagonized the suppressive effects of MUS, resulting in upregulation of salivary secretion. Quantitative RT-PCR and Western blotting revealed lesser expression of the PRG synthetase CYP11A1 protein and mRNA in the parotid, submandibular, and sublingual gland than in the cerebral cortex or adrenal gland as positive control organs. However, in response to methamphetamine withdrawal–induced stress, CYP11A1 production in each type of the salivary gland was highly upregulated to levels similar to those seen in the cerebral cortex. These results indicate that the salivary gland is capable of producing neurosteroids, as well as the brain. This suggests that steroid biosynthesis occurs in the salivary gland and is involved in the inhibitory regulation of salivary secretion in cooperation with GABA_A-R. Further studies are needed to determine the pathophysiological significance of the biosynthesis of neurosteroids and their mechanisms of action via nuclear and membrane receptors.