Abstract A study was made of the methyl sterols involved in sterol biosynthesis in developing rat brain. In addition to the determination of the endogenous sterols present in 15-day-old rat brain, experiments were conducted to establish which of these sterols could be labeled by a minced tissue preparation in the presence of [2-14C]mevalonic acid. The turnover of [14C]lanosterol, [4-3H]squalene oxide, and [14C]squalene by minced preparations was also shown. In the experiments involving incubation of labeled mevalonic acid with minced tissue preparations, the diunsaturated methyl sterols had a larger degree of labeling than the monounsaturated methyl sterols. Labeled squalene was also shown to be present in the mevalonate incubations. Under these in vitro conditions cholesterol was not found to be significantly labeled. Endogenous sterols of two types were examined, those occurring as steryl esters and those that were unesterified. A comparison of the distribution of sterols in these two fractions indicated a relatively small percentage of cholesterol (54.3%) in the steryl ester fraction. Based on gas chromatographic retention data and gas chromatography-mass spectrometry, the following sterols were identified: lanosterol, 4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol, 4,4-dimethyl-5α-cholest-8-en-3β-ol, 4α-methyl-5α-cholest-8-en-3β-ol, 4α-methyl-5α-cholesta-8,24-dien-3β-ol, 4α-methyl-5α-cholesta-7,24-dien-3β-ol, 4α-methyl-5α-cholest-7-en-3β-ol, 5α-cholesta-7,24-dien-3β-ol, desmosterol, and cholesterol.