Eucalyptus plantations have seldom responded to N fertilization in tropical and subtropical regions of Brazil. This implies that rates of N mineralization have been adequate to supply tree needs. However, subsequent crop rotations with low N fertilization may result in declining concentrations of organic and potentially mineralizable N (N0), and consequent loss of wood productivity. This study investigated (a) in situ N mineralization and N0 in soils of eucalypt plantations in São Paulo state, Brazil; (b) tree growth responses to N fertilizer applied 6–18 months after planting; and (c) the relationships between N0, other soil attributes and tree growth. We established eleven N fertilizer trials (maximum 240 kg ha−1 of N) in E. grandis and E. grandis x urophylla plantations. The soil types at most sites were Oxisols and Quartzipsamments, with a range of organic matter (18 to 55 g kg−1) and clay contents (8% to 67%) in the 0–20 cm layer. Concentrations of N0 were measured using anaerobic incubation on soil samples collected every three months (different seasons). The samples collected in spring and summer had N0 140–400 kg ha−1 (10%–19% total soil N), which were best correlated with soil texture and organic matter content. Rates of in situ net N mineralization (0–20 cm) ranged from 100 to 200 kg ha−1 year−1 and were not correlated with clay, total N, or N0. These high N mineralization rates resulted in a low response to N fertilizer application during the early ages of stand growth, which were highest on sandy soils. At the end of the crop rotation, the response to N fertilizer was negligible and non-significant at all sites.