The enzymatic properties as well as its distribution in the cerebral region and subcellular organells were investigated for the neurosteroid acyltransferase from the bovine brain, which synthesize the fatty acid esters of the neurosteroids. The cerebellum region was the highest in NSAT activity while the cerebrum was the lowest with 50% of the cerebellar activity. The NSAT was found to be mainly localized in the microsomal fraction. The optimal temperature and pH were 40℃ and 4.9, respectively. When ³H-DHEA was utilized as substrate, the K_m and V_(max) was 32.6 μM and 4.86 nmole/㎎ protein/h, respectively. Under the same condition pregnenolone(△^5P) was a competitive inhibitor with K₁=22.8 μM and testosterone was a uncompetitive inhibitor with K₁=28.2 μM. This may suggest that the NSAT has a different conformation in the acylation of the β-hydroxyl group at C-3 and C-17.