Incorporation of 13C from a dietary precursor into cholesterol was studied in neonatal rats. Rats were given uniformly 13C-enriched polyunsaturated fatty acids intragastrically and total lipid extracts of liver and brain were analyzed by 13C-NMR 1,4, 8, and 15 days later. 13C-enrichment was detected in brain but not in liver cholesterol. Maximal 13C-labeling was observed 4 days after injection of the label. Spectra revealed that 70% of newly incorporated 13C had 13C as an adjacent neighbor, the other 30% had 12C as the neighbor. Double quantum NMR revealed the arrangement in the cholesterol skeleton of the 13C-13C pairs transferred from precursors to cholesterol. Desmosterol, an intermediate of cholesterol synthesis, was identified in the spectra of brain lipids. Comparison of 13C-13C unit arrangements in both cholesterol and desmosterol allowed carbons 26 and 27 of desmosterol to be unambiguously assigned.