Abstract A field experiment was conducted to determine the effect of frequency of 15N tracer applications on the estimation of nitrogen (N) fixed by clover plants in an established mixed grass/clover pasture using the 15-enrichment technique. The results obtained showed that N derived from the atmosphere (%Ndfa) significantly decreased when 15N tracer was applied daily (87.2%) or at a 30-day interval (76.3%), after the first 30 days of regrowth. This effect did not persist at a further harvest, except on pastures treated with 150 kg N/ha of unlabelled fertiliser N. The estimate of the amount of N fixed was not significantly affected by the frequency of 15N applications but decreased from 36.7 to 19.8 kg N/ha after 60 days with fertiliser N application (150 kg N/ha). The mean daily rate of N fixation increased in the zero N treatment from 0.89 kg N/ha per day during the first 30 days regrowth to 1.36 kg N/ha per day during a subsequent 30 days of uninterrupted growth. Overall, the results showed that the 15N enrichment technique adequately estimated the amounts of clover N fixed in an established pasture under field conditions.