Recently proposed Regulatory Gate hypothesis states that mineralization of native soil organic matter is not regulated by the size, activity or composition of the soil microbial biomass.  This challenges one of the long held theories in soil microbiology that mineralization processes in soil are predominantly driven by microbial activities.  This study was conducted to test the Regulatory Gate hypothesis through evaluation of soil nitrogen (N) dynamics and mineralization by monitoring KCl-extractable N content in soils following a series of treatments, including air-drying, reweting and autoclaving, with or without subsequent laboratory incubations. The soils were from a century-old continuous winter wheat experiment with three treatments: unfertilized (CK), cattle manure addition once every four years (Manure), or treated with phosphorus (P) every year.  KCI-extractable N decreased with increasing incubation time in the unsterilized Manure soil, but remained fairly constant in all other soils tested.  Rewet of air-dried soils followed by 10 d incubation led to significant increase in the extractable N content . However, incubation for additional 10 d did not lead to the release of more extractable N (Check or sterilized Manure), or resulted in significant reduction of extractable N.  The highest extractable N detected was 23.4 mg N kg^-1 soil (or 3.55% of TN)  for Check and 43.5 mg N kg^-1 soil (or 5.3% of TN) for Manure.  The fact that extractable N content was highest following incubation of sterilized soils suggested that microbiological activities were not the key players in the mineralization of native soil organic matter.