PURPOSE: Metabolic imaging with hyperpolarized 13 C-labeled cell substrates is a promising technique for imaging tissue metabolism in vivo. However, the transient nature of the hyperpolarization, and its depletion following excitation, limits the imaging time and the number of excitation pulses that can be used. We describe here a single-shot three-dimensional (3D) imaging sequence and demonstrate its capability to generate 13 C MR images in tumor-bearing mice injected with hyperpolarized [1-13 C]pyruvate. METHODS: The pulse sequence acquires a stack-of-spirals at two spin echoes after a single excitation pulse and encodes the kz-dimension in an interleaved manner to enhance robustness to B0 inhomogeneity. Spectral-spatial pulses are used to acquire dynamic 3D images from selected hyperpolarized 13 C-labeled metabolites. RESULTS: A nominal spatial/temporal resolution of 1.25 × 1.25 × 2.5 mm3  × 2 s was achieved in tumor images of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate acquired in vivo. Higher resolution in the z-direction, with a different k-space trajectory, was demonstrated in measurements on a thermally polarized [1-13 C]lactate phantom. CONCLUSION: The pulse sequence is capable of imaging hyperpolarized 13 C-labeled substrates at relatively high spatial and temporal resolutions and is robust to moderate system imperfections. Magn Reson Med 77:740-752, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.