Methylglyoxal (MG), a metabolic by-product, reacts with certain proteins to yield irreversible advanced glycation end products (AGEs) and increases oxidative stress that causes the pathophysiological changes in diabetes, hypertension, and aging. Although MG production from glucose has been well documented, the contribution of other intermediates of different metabolic pathways to MG formation is far less known. Our aim was to determine and compare the formation of MG, MG-induced AGE, N(epsilon)-carboxyethyl-lysine (CEL), inducible nitric oxide synthase (iNOS), nitric oxide, and peroxynitrite from different metabolic precursors in cultured rat aortic vascular smooth muscle cells (VSMCs). High-performance liquid chromatography was used to determine MG levels, whereas nitrite + nitrate, indicators of nitric oxide production, and peroxynitrite levels were measured with specific assay kits. The CEL and iNOS were detected using immunocytochemistry. There was a concentration-dependent increase in MG levels in VSMCs after 3-hour incubation with 5, 15, and 25 mmol/L of D-glucose, fructose, or aminoacetone. Aminoacetone produced a 7-fold increase in MG levels above the basal value followed by fructose (3.9-fold), D-glucose (3.5-fold), acetol (2.8-fold), and sucrose (2.3-fold) after a 3-hour incubation with 25 mmol/L of each precursor. L-Glucose, 3-O-methylglucose, and mannitol had no effect on MG production. All precursors, except l-glucose, 3-O-methylglucose and mannitol, increased CEL. Aminoacetone, D-glucose, and fructose significantly increased iNOS, nitrite/nitrate, and peroxynitrite levels. In conclusion, aminoacetone is the most potent precursor of MG production in VSMCs, followed by fructose and d-glucose. This could have important implications in relation to high dietary fructose and protein intake.