Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-(13)C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-(13)C] pyruvate were even longer at 3 T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-(13)C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-(13)C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-(13)C] pyruvate or polarized [1-(13)C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-(13)C]pyruvic acid, 13C sodium bicarbonate, [1,4-(13)C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.