Pregnenolone (P) and its sulfate ester (PS) have been characterized in the brain of adult male rats. The concentration of P (38.4 +/- 6.9 and 22.1 +/- 2.9 ng/g, mean +/- S.D., in anterior and posterior brain, respectively) exceeded that of PS in brain (15.8 +/- 3.0 and 5.7 +/- 2.1 ng/g in the same fractions) and largely those of P and PS in plasma (1.3 +/- 0.2 and 1.4 +/- 0.3 ng/g, respectively). The level of P in brain was much larger than that of dehydroepiandrosterone sulfate (DS), characterized and measured previously (Corpéchot et al.). Brain P and PS levels did not seem to depend on steroidogenic gland secretion: no meaningful difference occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. It is proposed that, as that of DS (ref. 5) P and PS formation or accumulation (or both) in the rat brain depend on in situ mechanisms unrelated to the peripheral endocrine gland system.