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The spatial distribution of organic substrates and microscale soil heterogeneity significantly influence organic matter (OM) persistence as constraints on OM accessibility to microorganisms. However, it is unclear how changes in OM spatial heterogeneity driven by factors such as soil depth affect the relative importance of sub-strate spatial distribution on OM persistence. This work evaluated the decomposition and persistence of 13C and 15N labeled water-extractable OM inputs over 50 days as either hotspot (i.e., pelleted in 1-2 mm-size pieces) or distributed (i.e., added as OM < 0.07 mu m suspended in water) forms in topsoil (0-0.2 m) and subsoil (0.8-0.9 m) samples of an Andisol. We observed greater persistence of added C in the subsoil with distributed OM inputs relative to hotspot OM, indicated by a 17% reduction in cumulative mineralization of the added C and a 10% higher conversion to mineral-associated OM. A lower substrate availability potentially reduced mineralization due to OM dispersion throughout the soil. NanoSIMS (nanoscale secondary ion mass spectrometry) analysis identified organo-mineral associations on cross-sectioned aggregate interiors in the subsoil. On the other hand, in the topsoil, we did not observe significant differences in the persistence of OM, suggesting that the large amounts of particulate OM already present in the soil outweighed the influence of added OM spatial distribution. Here, we demonstrated under laboratory conditions that the spatial distribution of fresh OM input alone significantly affected the decomposition and persistence of OM inputs in the subsoil. On the other hand, spatial distribution seems to play a lower role in topsoils rich in particulate OM. The divergence in the influence of OM spatial distribution between the top and subsoil is likely driven by differences in soil mineralogy and OM composition. Technical University of Munich Institute for Advanced Study; NSF [DMR-1654596]; Packard Foundation; NSF MRSEC program [DMR-1719875]; Kavli Institute at Cornell; DOE EFRC BES [DE-SC0001086, NSF-MRI-1429155]; Cornell University; Weill Institute; KIC Published version The Technical University of Munich Institute for Advanced Study provided funding for this study. MJZ and LFK acknowledge support by the NSF (DMR-1654596) and Packard Foundation. This work used the Cornell Center for Materials Research Shared Facilities supported through the NSF MRSEC program (DMR-1719875). Additional support for the FIB/SEM cryo-stage and transfer system was provided by the Kavli Institute at Cornell (KIC) for Nanoscale Science and the Energy Materials Center at Cornell, DOE EFRC BES (DE-SC0001086). The FEI Titan Themis 300 was acquired through NSF-MRI-1429155, with additional support from Cornell University, the Weill Institute, and the KIC. The authors thank Katherine E. Grant and Louis A. Derry (Cornell University Earth and Atmospheric Sciences) for providing soil samples from the Polulu Flow, HI. The authors thank Akio Enders and Kelly Hanley for their technical support.