This study examines the role of nitrogen transformations in the acidification of soil under stock urine patches, specifically the formation of acidic subsurface layers. These are horizontal planes of acidity several centimetres below the soil surface. Glasshouse studies were conducted to relate nitrogen transformations to measured pH changes in soil treated with urine or urea solution (simulated urine). Acidic subsurface layers formed in both urine- and simulated urine-treated soil. With the development of a H+ balance model, the contribution of nitrogen transformations to changes in the H+ concentrations in simulated urine patches was determined. During the first 9 days following treatment, urea hydrolysis and NH3 volatilisation dominated changes in H+ concentration. After that, net immobilisation contributed to H+ changes; however, nitrification was the dominant process occurring. Downward movement of NH4+ originating from urea hydrolysis allowed more nitrification to occur in lower soil layers. The net result of these processes was net acidification of the 4'6, 6'8, and 8'10 cm layers by approximately 0.7, 0.6, and 0.3 pH units, respectively. Thus nitrogen transformations were responsible for the formation of acidic subsurface layers in simulated stock urine patches within 6 weeks of application.