This study examined the influence of simulated urine solutions containing various KCl and urea-N rates on the formation of acidic subsurface layers in soil columns. A factorial design was implemented with application rates equivalent to 0, 21, 42, 63, and 84 g urea-N/m2 and 0, 12.5, 25, and 37.5 g KCl-K/m2. The addition of N caused the formation of acidic subsurface layers at depths between 0.02 and 0.10 m. The magnitude of the resultant net acidification and the depth of the most acidic layer increased with N rate. More acidification occurred at depth at the higher N rates due to the downward movement of NH4+-N. The inclusion of K in the simulated urine produced less acidity in the surface layers and more acidity at depth as the K application rate increased owing to competition between K+ and NH4+-N for exchange sites, allowing more NH4+-N to move to depth. The residual acidity in the soil at the completion of the experiment was found to be greater than the alkalinity of plant material. Therefore, acidic subsurface layers are likely to persist after plant death and decomposition. We also examined the impact of defoliation on the resultant pH profiles formed following simulated urine addition. Defoliation accentuated the magnitude of acidic subsurface layers, possibly due to changes in the rate of N uptake. The influence of defoliation was minor compared with the main effects of N addition.