Under rainforest vegetation, the central Amazonian Ferralsols are characterized by relatively high availability of N in relation to other nutrients. After forest clearing, several tree crops also have not shown yield responses to N fertilizer. To elucidate the mechanisms of this apparent N sufficiency, the mineralization of soil N was measured under three tree crops and a leguminous cover crop (Pueraria phaseoloides) in a multi-strata agroforestry system at two fertilizer input levels on a Xanthic Ferralsol in central Amazonia. In situ incubations of topsoil (0–10 cm) were carried out using the buried-bag method on five occasions over ten months. The highest mineralization rates were found under the cover crop, intermediate rates under rubber trees (Hevea brasiliensis) where the soil was also covered by the cover crop, and lowest rates under peach palm (Bactris gasipaes) and cupuaçu (Theobroma grandiflorum) with no cover crop. The increased N mineralization under the cover crop was due to more total N in the soil, higher soil moisture and, presumably, a larger pool of readily mineralizable soil N compared with the soil under the tree crops. Other fertility parameters also differed significantly between sampling positions within the plots, but this had no major influence on net N mineralization. Also, the input of NPK fertilizer and dolomite had no significant influence on N mineralization, indicating that N mineralization was not nutrient-limited. High total N mineralization rates in the soil (approximately 350 kg ha−1 a−1 at 0–10 cm depth) explained earlier observations of nitrate leaching into the subsoil under multi-strata agroforestry at this site. Considering the spatial patterns of N mineralization with maximum values under the cover crop, the exploration of the soil volume by crop roots should be maximized to increase the uptake of mineralized soil N by the crops and reduce nitrate leaching. Appropriate measures are narrow tree spacing, use of annual and semi-perennial intercrops and encouragement of the lateral root development of the trees. In addition, the mineralization of soil N close to the tree crops can be influenced through the management of the cover crop. In view of the high total N mineralization rates in the system and unclear yield responses of tree crops to N fertilizer, the application of N fertilizer to tree crops with well-developed root systems and a well-managed cover crop may often be unnecessary on this soil type. This may facilitate the further development of tree crop agriculture in the region.