PURPOSE: To compare carbon-13 ((13) C) MRSI of hyperpolarized [1-(13) C]pyruvate metabolism in a murine tumor model with mass spectrometric (MS) imaging of the corresponding tumor sections in order to cross validate these metabolic imaging techniques and to investigate the effects of pyruvate delivery and tumor lactate concentration on lactate labeling. METHODS: [1-(13) C]lactate images were obtained from tumor-bearing mice, following injection of hyperpolarized [1-(13) C]pyruvate, using a single-shot 3D (13) C spectroscopic imaging sequence in vivo and using desorption electrospray ionization MS imaging of the corresponding rapidly frozen tumor sections ex vivo. The images were coregistered, and levels of association were determined by means of Spearman rank correlation and Cohen kappa coefficients as well as linear mixed models. The correlation between [1-(13) C]pyruvate and [1-(13) C]lactate in the MRS images and between [(12) C] and [1-(13) C]lactate in the MS images were determined by means of Pearson correlation coefficients. RESULTS: [1-(13) C]lactate images generated by MS imaging were significantly correlated with the corresponding MRS images. The correlation coefficient between [1-(13) C]lactate and [1-(13) C]pyruvate in the MRS images was higher than between [1-(13) C]lactate and [(12) C]lactate in the MS images. CONCLUSION: The inhomogeneous distribution of labeled lactate observed in the MRS images was confirmed by MS imaging of the corresponding tumor sections. The images acquired using both techniques show that the rate of (13) C label exchange between the injected pyruvate and endogenous tumor lactate pool is more correlated with the rate of pyruvate delivery to the tumor cells and is less affected by the endogenous lactate concentration.