Purpose: The aim of this study was to investigate the change in signal sensitivity over different acquisition start times and optimize the scanning window to provide the maximal signal sensitivity of [1-13C]pyruvate and its metabolic products, lactate and alanine, using spatially localized hyperpolarized 3D 13C magnetic resonance spectroscopic imaging (MRSI). Materials and Methods: We acquired 3D 13C MRSI data from the brain (n = 3), kidney (n = 3), and liver (n = 3) of rats using a 3T clinical scanner and a custom RF coil after the injection of hyperpolarized [1-13C]pyruvate. For each organ, we obtained three consecutive 3D 13C MRSI datasets with different acquisition start times per animal from a total of three animals. The mean signal-to-noise ratios (SNRs) of pyruvate, lactate, and alanine were calculated and compared between different acquisition start times. Based on the SNRs of lactate and alanine, we identified the optimal acquisition start timing for each organ. Results: For the brain, the acquisition start time of 18 s provided the highest mean SNR of lactate. At 18 s, however, the lactate signal predominantly originated from not the brain, but the blood vessels; therefore, the acquisition start time of 22 s was recommended for 3D 13C MRSI of the rat brain. For the ki...