Abstract: Estimating soil N mineralization is important for determining the amount of N fertilizer needed to obtain optimum yields at minimal environmental and economic costs. The aim of this work was to determine the most appropriate laboratory method for the estimation of N mineralization during a winter wheat-growing season in calcareous soils under a humid Mediterranean climate. Laboratory methods were developed involving three chemical extractants, CaCl2, KCl, and NaHCO3, and several soil-drying and extraction temperatures. Soil N indexes calculated based on extractions were compared with potentially mineralizable N (No). Moreover, soil mineralization indexes estimated from both chemical extractions and aerobic incubation were related to apparent N mineralization and wheat N uptake in a pot experiment. The mineralization index estimated from an extraction with KCl boiled at 100°C (HotKCl) was the index that best correlated with No and the apparent mineralization under greenhouse conditions. The combination of preplant soil mineral N and the HotKCl N mineralization index was more strongly correlated with wheat N uptake than soil mineral N data alone. Consequently, estimation of N mineralization using HotKCl extraction is the most appropriate methodology for establishing N fertilizer use recommendations for wheat cultivation in calcareous soils under Mediterranean conditions.