Studies of fatty acid (FA) esterification by adipocytes have led to conflicting views with respect to how the process is regulated by norepinephrine (NE). It remains unclear whether NE directly modulates the pathway or whether its effects are indirect and reflect its well-known action to activate lipolysis. Changes in lipolysis can complicate estimation of esterification rates by altering both medium FA and the hydrolysis of newly formed FA esters. In this report, we describe an experimental approach that determined the effect of NE on FA esterification, amidst the complications introduced by activation of lipolysis. Esterification rates were estimated from the simultaneous incorporations (0.1-60 min) of [14C]glucose and [3H]oleate into diglyceride (DG), phospholipid (PL), and triglyceride (TG). Saturation kinetics of incorporation rates, with respect to FA, and more specifically to unbound or albumin-free FA (ubFA), were determined in both basal and NE-treated cells. To obtain true estimates of ester synthesis, incorporation rates were adjusted for label loss from breakdown of labeled esters. Our findings were: 1) In basal versus NE-treated cells, [3H]oleate, on its pathway to esterification, was diluted, respectively, by 2 and 50% of measured cell FA, and the diluting FA appeared derived from lipolysis. 2) Syntheses of PL, DG, and TG, estimated from incorporation of [14C]glucose, saturated at low ubFA. The Km for TG synthesis (0.06 microM) was within the physiological range of ubFA which meant that changes in plasma FA will modulate TG synthesis. PL synthesis, on the other hand (Km less than 0.01 microM), would be largely saturated under physiological conditions. 3) NE treatment increased the molar ratio of FA to albumin in the medium an average 8-fold and ubFA about 87-fold. In addition, NE accelerated hydrolysis of labeled PL and DG. Adjusting incorporation rates for these changes indicated that NE does not directly regulate glyceride synthesis. The assays described should allow estimation of glycerolipid synthesis under various metabolic or disease states and will distinguish direct effects from those reflecting changes in FA concentration or in hydrolysis of labeled FA esters.