Fractional lipid synthesis can be measured using the incorporation of deuterium from deuterated water. The calculations require knowledge of the maximum incorporation number (N) of deuterium atoms in the molecules synthesized. For both tissue palmitate and cholesterol, N values have been found to be higher during in vivo versus in vitro experiments. We determined the N values to be used for measuring the fractional synthesis of plasma cholesterol and of palmitate triglycerides (TG). Rats were given drinking water enriched (7% to 10%) with deuterated water, and N was determined from the mass isotopomer distributions of plasma cholesterol and plasma TG palmitate and the deuterium enrichment of plasma water. We found N to be 21 for palmitate and 27 for cholesterol. These values agree with those reported for tissue palmitate and cholesterol in vivo, and are higher than values found in vitro. We also found large deuterium enrichments in plasma glucose and in liver lactate and pyruvate. We suggest that, compared with in vitro studies, in vivo metabolism of these compounds leads to an additional pathway of incorporation of deuterium into lipids through deuterium-labeled acetyl coenzyme A (CoA). This could explain why N values are higher in vivo than in vitro.