The enzymatic pathway for synthesis of fatty acids from acetyl-coenzyme A, or de novo lipogenesis (DNL), is present in human liver and, to a lesser extent, in adipose tissue. Although the molecular and enzymatic regulation of the components for DNL are well characterized, the quantitative importance of the assembled pathway and its physiologic functions have remained uncertain. We review methods that have been used for measuring DNL in vivo, their limitations and the conclusions based on them. Two new methods for direct measurement of DNL in humans are discussed-mass isotopomer distribution analysis (MIDA), a mass spectrometric technique based on combinatorial probabilities, and 2H2O incorporation. Recent findings with these methods in a variety of dietary and hormonal settings are reviewed. In particular, we focus on the question of whether or not surplus carbohydrate energy is converted to fat by the liver in humans. A somewhat surprising model of the response to carbohydrate over-feeding emerges from these studies, with a number of implications for metabolic regulation in health and disease. We close by speculating on potential functions of DNL in physiology and pathophysiology if storage of surplus carbohydrate energy is not an important function of DNL. The availability of techniques for quantifying DNL in vivo should make it possible to resolve these uncertainties regarding its functions and regulation in humans.