A field study was conducted to investigate the effects of N fertilization on soil N pools and associated microbial properties in a 13-year-old hoop pine (Araucaria cunninghamii) plantation of southeast Queensland, Australia. The treatments included: (1) control (without N application); (2) 300 kg N ha-1 applied as NH4NO3; and (3) 600 kg N ha-1 as NH4NO3. The experiment employed a randomized complete block design with four replicates. Soil samples were taken approximately 5 years after the N application. The results showed that application of 600 kg N ha-1 significantly increased concentrations of NH4+-N in 0-10 cm soil compared with the control and application of 300 kg N ha-1. Concentrations of NO3--N in soil (both 0-10 cm and 10-20 cm) with an application rate of 600 kg N ha-1 were significantly higher compared with the control. Application of 600 kg N ha-1 significantly increased gross N mineralization and immobilization rates (0-10 cm soil) determined by 15N isotope dilution techniques under anaerobic incubation, compared with the control. However, N application did not significantly affect the concentrations of soil total C and total N. N application appeared to decrease microbial biomass C and N and respiration, and to increase the metabolic quotient (qCO2) in 0-10 cm soil, but these effects were not statistically significant. The lack of statistical significance in these microbial properties between the treatments might have been associated with large spatial variability between the replicate plots at this experimental site. Spatial variability in soil microbial biomass C and N was found to relate to soil moisture, total C and total N.