Measurements of hyperpolarized ¹³C label exchange between injected [1-¹³C]pyruvate and the endogenous tumor lactate pool can give an apparent first order rate constant for the exchange. Determination of isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measuring tumor uptake of [1-¹⁴C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first order rate constant for hyperpolarized ¹³C label exchange gave an isotope flux that showed good agreement with a flux determined directly by injecting non polarized [3-¹³C]pyruvate and then rapidly excising the tumor after 30 s and measuring ¹³C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measuring labeled lactate concentration in blood and tumor, and by examining the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1-¹³C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor but that labeled lactate in the blood pool produced by injection of hyperpolarized [1-¹³C]pyruvate has only relatively low levels of polarization. This study has shown that measurements of hyperpolarized ¹³C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined.