Pregnenolone sulfate (PREGS) is reported to be present in higher concentration in the brain (more than 5 ng/g tissue in the rat) than in blood and is considered to be a neurosteroid. However, there are some doubts on its brain levels, because they were determined by indirect methods (e.g., GC-MS or radioimmunoassay after solvolysis). In the present study, PREGS in rat brains was determined by an enzyme-linked immunosorbent assay, which did not require solvolysis, after pretreatment with an Oasis HLB cartridge. The absolute recovery rate of PREGS through the pretreatment was 60.8%, and the quantitation limit was 33 pg/g tissue for a 200-mg of brain aliquot. Intra- and inter-assay coefficients of variation were less than 15.1 and 9.2%, respectively. The brain PREGS levels in the control rats (n=10) were less than 0.15 ng/g tissue except for one sample (0.42 ng/g tissue) and were lower than the serum levels (n=5, 0.25—0.47 ng/ml). On the contrary, the brain PREGS levels were sufficiently increased after intrapertioneal injection of 2 mg/kg body of PREGS (n=5, 0.37—1.29 ng/g tissue). These results demonstrate that, in rats, the brain PREGS may be derived from peripheral sources, and its actual levels are much lower than those previously measured by indirect methods.