The metabolite flux of carbohydrates through primary catabolism and into hexose resynthesis has been investigated for alginic acid biosynthesis in Azotobacter vinelandii. To do these studies, we fed the microorganism a variety of 13C-labeled glucose precursors, including [U-13C6]glucose. The incorporations of the precursors were determined by 1D 13C-NMR, 2D 13C-DQF-COSY, and inverse triple-quantum correlation experiments. The results clearly show that the entire catabolism of hexose is through the Entner-Doudoroff (E-D) pathway and that the triose pools are in equilibrium. Reentry into gluconeogenesis prior to alginate synthesis occurs totally from the glyceraldehyde 3-phosphate generated by the E-D pathway. The obligatory intermediacy of triose intermediates in alginate biosynthesis was proved. The experiments and results presented in this paper constitute a new method for distinguishing the E-D pathway from glycolysis in bacteria.