This study aimed to ascertain whether application of sheep urine led to the development of acidic subsurface layers of a pasture soil. Deionised water or simulated urine solution delivering urea-N at 44.8 g m-2 and K+ at 25 g m-2 was applied to soil in either winter or spring. Treatments were applied to the soil surface within 10.3 cm internal diameter PVC tubes inserted 20 cm into the soil either under ryegrass or kept bare. Main sampling times corresponded to the completion of various soil N transformations as determined by periodic sampling. Main samplings involved the collection of above ground plant material and soil sampling in 2 cm depth increments in 0-10 cm and 5 cm intervals in 10-20 cm depths. Following treatment application, Urea and NH4+-N moved to a depth no greater than 20 cm but the extent of movement was greater in winter than spring due to the influence of initial soil moisture. Following urea hydrolysis, soil pH increased in the 0-15 cm depth. Subsequent nitrification significantly acidified soil under pasture by 0.8 to 1.0 pH units in the 2-8 and 2-6 cm depths in winter and spring, respectively. This created a net acidic subsurface layer of 0.2-0.4 pH units compared with soil at the beginning of the experiment. Subsurface acidification was 0.5-0.7 pH units greater in bare soil compared with the presence of pasture. Transformations of N resulting from application of simulated urine solution formed acidic subsurface layers in the field regardless of the season of application.