Nitrification and nitrate leaching have been suggested to be major causes of soil acidification. However, it is unknown whether these processes cause subsoil acidification. Soil column experiments examined the effect of the addition of Ca(NO3)(2) or (NH4)(2)SO4 to the topsoil horizon on subsoil acidification under nodulated lupins (Lupinus angustifolius L.) and subterranean clover (Trifolium subterraneum L.). Nitrate leaching was achieved by adding excess water to the surface of the columns.Where basal nutrients were applied only to the top 10-cm layer, about 60-70% of the total root length of lupin and over 50% of subterranean clover were distributed in that layer. Plants grown without added nitrate for 105 days decreased soil pH at all layers but more significantly in the top 20 cm (by up to 0.7 units); the decrease in pH correlated well with increased root length density of both species (r(2) = 0.98, n = 9). The addition of Ca(NO3)(2) to the top 10-cm layer caused less acidification by about 0.1 pH units at all depths than the treatment without Ca(NO3)(2).Where basal nutrients were applied uniformly throughout the column, root length density of lupin and subterranean clover tended to increase with depth. The addition of (NH4)(2)SO4 in the top 10 cm significantly increased NO3- concentration in all layers but NH4+ was mainly retained in the top 30-cm layer. Lupin and subterranean clover grown without added NH4+ for 82 days decreased soil pH by 0.3 units at all depths. Compared with the plants receiving no (NH4)(2)SO4, lupin grown with (NH4)(2)SO4 at 0-10 cm depth in the column caused more acidification by 0.05-0.2 pH units in the top 10 cm but less acidification by 0.15-0.17 units at 10-40 cm depth in the column; subterranean clover grown with (NH4)(2)SO4 caused more acidification by 0.35-0.46 units in the top 10 cm and less acidification by 0.14-0.19 units in the 20-50 cm layer.The results suggest that the leaching of nitrate from topsoil is unlikely to cause subsoil acidification. In contrast, the uptake of nitrate by the roots reduces net acid production in subsoil layers.