Herein we report a study combining metabolomics and mass isotopomer analysis used for investigation of the biochemical fate of γ- hydroxybutyric acid (GHB). Using various 13C incorporation labeling patterns into GHB, we have discovered that GHB is catabolized by previously unknown processes that include (i) direct β-oxidation to acetyl-CoA and glycolate, (ii) α-oxidation to 3-hydroxypropionyl-CoA and formate, and (iii) cleavage of C-4 to yield 3-hydroxypropionate and CO2. We further utilized the unique attributes of our labeling patterns and the resultant isotopomers to quantitate relative flux down the identified pathways. © 2014 American Chemical Society.