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The nitrogen fixation ability of Trifolium vesiculosum cv. Amclo and Trifolium subterraneum cv. Mount Barker in pure and mixed stands with Lolium multiflorum cv. Midmar were compared using the nitrogen difference method (NDM). The percentage and quantity of apparent legume?derived nitrogen, as affected by different nitrogen applications and grass seeding rates, was estimated. The response of both clover species varied with treatments in pure and mixed stands. The apparent legume?derived nitrogen ranged from 15?98% and from 43?94%, while quantities ranged from 20?102 kg ha−1 and from 68?260 kg ha−1 for T. vesiculosum and T. subterraneum, respectively. Results indicate that the uptake of available soil nitrogen contributed to the total nitrogen content of both clover species. However, since the application of the NDM is limited, conclusions as to the response differences and the possible transfer of nitrogen to the grass component could be unreliable.