Incorporation of [3H]oleate and [14C]glucose into cellular lipids was studied in the preadipose cell line BFC-1 to determine flux changes that accompany the adipose conversion process. Dilution of oleate by intracellular fatty acids (FA) was estimated from the 3H/14C incorporation ratios and from relating steady-state radioactivity in diglycerides to their measured cellular levels. The data indicated that exogenous FA mixed with less than 1% of endogenous FA on its pathway to esterification. Conversion of preadipocytes to adipocytes increased uptake of FA and glucose by approximately 3-fold and synthesis of diglycerides and triglycerides by 5- and 16-fold, respectively, with little if any increase of phospholipid synthesis. A 50% drop in 3H/14C incorporation ratio indicated a doubling of the rate at which endogenous FA mixed with the exogenous FA that had entered the cell. Adipocytes compared with preadipocytes exhibited a 50% greater cell diameter and a doubling of intracellular water volume and of protein and phospholipid levels, reflecting cellular enlargement consequent to the arrest of cell division that precedes adipose conversion. Diglyceride levels were also increased in adipocytes, however, since their turnover was fast, as indicated by rapid equilibration of diglyceride labeling; the increase reflected changes in their relative rates of synthesis and disposal. Diglyceride levels related to cell phospholipid, and other indexes of cell size remained constant. This indicated that the supply of diglycerides was tightly coupled to the synthesis of triglycerides and phospholipids, which suggested feedback regulation of diglyceride formation. The studies provide a methodological approach to measurement and interpretation of rates of lipid deposition in cultured cells.