The application of MS to imaging, or MS imaging (MSI), allows for the direct investigation of tissue sections to identify biological compounds and determine their spatial distribution. We present an approach to MSI that combines secondary ion MS (SIMS) and MALDI MS for the imaging and analysis of rat spinal cord sections, thereby enhancing the chemical coverage obtained from an MSI experiment. The spinal cord is organized into discrete, anatomically defined areas that include motor and sensory networks composed of chemically diverse cells. The MSI data presented here reveal the spatial distribution of multiple phospholipids, proteins, and neuropeptides obtained within single, 20 μm sections of rat spinal cord. Analyte identities are initially determined by primary mass match and confirmed in follow-up experiments using LC MS/MS from extracts of adjacent spinal cord sections. Additionally, a regional analysis of differentially localized signals serves to rapidly screen compounds of varying intensities across multiple spinal regions. These MSI analyses reveal new insights into the chemical architecture of the spinal cord and set the stage for future imaging studies of the chemical changes induced by pain, anesthesia, and drug tolerance.