AbstractPlant nutrition requires organic nitrogen to be mineralized before roots can absorb it. A 13‐year field study was conducted on typical rain‐fed Mediterranean Vertisol to determine the effects of tillage system, crop rotation and N fertilizer rate on the long‐term NH4+–N content in the soil profile (0–90 cm). The experiment was designed as a randomized complete block with a split–split plot arrangement and three replications. The main plots tested the effects from the tillage system (no‐tillage and conventional tillage); the subplots tested crop rotation with 2‐year rotations (wheat–wheat, wheat–fallow, wheat–chickpea, wheat–faba bean and wheat–sunflower) and the sub‐subplots examined the N fertilizer rate (0, 50, 100 and 150 kg N/ha). Soil NH4+–N content was greatest in the rainiest years and greater under the no‐tillage (NT) system than the conventional tillage (CT) system (57 and 48 kg/ha, respectively). The deepest soil (30–60 and 60–90 cm) contained a greater NH4+–N content (21.0 and 21.4 kg/ha, respectively) than the shallowest soil (19.5 kg/ha in 0–30 cm). This observation may be related to Vertisol characteristics, especially crack formation that allows greater mineralization in the deepest layers by displacing organic matter.