In this study, the three-dimensional spatial distributions of a number of metabolites involved in regulating symbiosis and biological nitrogen fixation (BNF) within soybean root nodules were revealed using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While many metabolites exhibited distinct spatial compartmentalization, some metabolites were asymmetrically distributed throughout the nodule (e.g., S-adenosylmethionine). These results establish a more complex metabolic view of plant-bacteria symbiosis (and BNF) within soybean nodules than previously hypothesized. Collectively these findings suggest that spatial perspectives in metabolic regulation should be considered to unravel the overall complexity of interacting organisms, like those relating to associations of nitrogen-fixing bacteria with host plants.