Steroid intermediates of the cholesterol synthesis pathway are characterized by rapid turnover rates relative to cholesterol due to their small pool size. Because the small pools will label rapidly, these intermediates may provide valuable information about the incorporation of isotopes in de novo synthesis of cholesterol and related compounds. The labeling of cholesterol synthesis intermediates from [1-¹³C]acetate was investigated in human subjects and in liver cell models by means of isotopomer spectral analysis (ISA). In human subjects, infusing [1-¹³C]acetate into the duodenum for 12 h demonstrated that ~50% of the plasma lathosterol pool was derived from de novo synthesis during this interval. The lipogenic acetyl-CoA precursor pool enrichment reached a constant value within 3 h of the start of the infusion. In vitro studies indicated that liver cell models decrease de novo lathosterol synthesis when cholesterol synthesis is inhibited by statins or cholesterol-containing serum. We propose a new calculation to increase the accuracy and precision of cholesterol synthesis estimates in vivo combining the ISA of lathosterol and cholesterol.