Steroid intermediates of the cholesterol synthesis pathway are characterized by rapid turn-over 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 using Isotopomer Spectral Analysis (ISA). In human subjects, infusing [1-¹³C]acetate into the duodenum for 12 hours demonstrated that approximately 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 three hours of starting the infusion. In vitro studies indicated that liver cell models decrease do 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 analysis of lathosterol and cholesterol.