Nitrate (NO3−) and ammonium (NH4+) are the main inorganic nitrogen sources available to plants. However, exclusive ammonium nutrition may lead to stress characterized by growth inhibition, generally associated with a profound metabolic reprogramming. In this work, we investigated how metabolism adapts according to leaf position in the vertical axis of tomato (Solanum lycopersicum cv. M82) plants grown with NH4+, NO3−, or NH4NO3 supply. We dissected leaf biomass composition and metabolism through an integrative analysis of metabolites, ions, and enzyme activities. Under ammonium nutrition, carbon and nitrogen metabolism were more perturbed in mature leaves than in young ones, overall suggesting a trade-off between NH4+ accumulation and assimilation to preserve young leaves from ammonium stress. Moreover, NH4+-fed plants exhibited changes in carbon partitioning, accumulating sugars and starch at the expense of organic acids, compared with plants supplied with NO3−. We explain such reallocation by the action of the biochemical pH-stat as a mechanism to compensate the differential proton production that depends on the nitrogen source provided. This work also underlines that the regulation of leaf primary metabolism is dependent on both leaf phenological stage and the nitrogen source provided.Read less <