The characterization and quantification of pregnenolone in human sciatic nerves were undertaken, following previous demonstration of the synthesis of this steroid in rat brain oligodendrocytes, to explore the hypothesis that Schwann cells may demonstrate the same biosynthetic activity. Pregnenolone was definitively identified by mass spectrometry and quantified by specific radioimmunoassay. Its concentration (mean +/- SD, 63.9 +/- 45.9 ng/g of wet tissue, n = 12) was greater than or equal to 100 times the plasma level and concentration found in tendons and muscle. No correlation was found with sex or age. Free dehydroepiandrosterone as well as sulfate and fatty acid esters of pregnenolone and dehydroepiandrosterone were also measured. Results are discussed in terms of the concept that these "neurosteroids" may be synthesized in the peripheral nervous system.