Pregnenolone is an endogenous steroid hormone involved in the biological synthesis of other hormones. Pregnenolone is monitored as an ancillary test for congenital adrenal hyperplasia (CAH), particularly in situations where diagnosis of 21-hydroxylase and 11-hydroxylase deficiency has been ruled out. This stable labeled internal standard is suitable for numerous LC/MS and GC/MS applications in endocrinology, clinical chemistry, sports testing, and isotope dilution methods.