The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into 14CO2 and 14C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that of D-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with 14C showed that more carbon from fructose than from glucose was incorporated into CO2 and glycerolipids. The relative distribution of 14C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures.