A major product of mitochondrial and peroxisomal -oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal -oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood, and are the subjects of our research. In this report, we describe a study of -oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-¹³C₁₈]stearate or [U-¹³C₁₆]palmitate. Using mass isotopomer analysis, we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the TCA cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mM [U-¹³C₁₈]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mM [U-¹³C₁₆]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that (1) chain elongation activity is a useful and novel probe for peroxisomal -oxidation and (2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.