Abstract Ammonium-fed plants may acidify the rhizosphere and thus increase availability of Mn in calcareous alkaline soils. The importance of N nutrition in the differential expression of tolerance to Mn deficiency among cereal genotypes is not yet clear. Two factorial experiments testing effects of the NH 4-N/NO 3-N ratio and Mn fertilization on growth of barley genotypes differing in tolerance to Mn deficiency were conducted in two calcareous alkaline soils in pots in a controlled environment. In the soil containing 80% CaCO 3at pH 8.5, better root and shoot growth and higher shoot Mn concentrations were achieved with nitrate supply, especially at lower rates of Mn fertilization. The Mn-efficient genotype Weeah (tolerant of Mn deficiency) achieved better root and shoot growth than Mn-inefficient Galleon barley (sensitive to Mn deficiency) regardless of experimental treatment. Fertilization with Mn did not influence total N concentration in barley roots and shoots. In the soil containing 5% CaCO 3at pH 7.8, ammonium-fed plants had better root and shoot growth and, at shoot Mn concentrations above the critical level, Mn-inefficient Galleon performed better than Mn-efficient Weeah barley. It appears that differential expression of Mn efficiency among barley genotypes is not associated with differences in Mn availability expected to be produced by differential rhizosphere acidification as a response to different forms of N supply. There is an apparent preference of locally selected barley genotypes for nitrate nutrition when grown on the highly calcareous alkaline soils of southern Australia.