A field experiment was performed in eastern Kenya to estimate N2 fixation by Sesbania sesban over an 18-month period using the 15N dilution method. The influence of three reference species, Senna spectabilis, Eucalyptus saligna and Grevillea robusta, on the estimates of N2 fixation was also assessed. Percentage Ndfa (nitrogen derived from the atmosphere) was calculated based on foliar atom excess (FAE), above-ground atom excess (AAE) or whole tree atom excess (WAE) data. The differences in atom% 15N excess values between species and plant parts are presented and discussed. We recommend the use of several reference species for estimating %Ndfa and that the different results obtained should be carefully considered in relation to the issues being addressed. In this study, Senna was the most suitable of the three reference species because its N uptake pattern and phenology were very similar to those of Sesbania. When well established, the amount of N fixed by Sesbania accounts for more than 80% of its total N content, according to FAE-based estimates. We estimated the Ndfa by Sesbania after 18 months to between 500 and 600 kg ha?1 , depending on whether FAE, AAE or WAE data were used and on the choice of reference species. The substantial accumulation of N in planted Sesbania highlighted its potential to increase the sustainability of crop production on N-limited soils. We consider the 15N dilution method to be appropriate for quantifying N2 fixation in improved fallows in studies, similar to this one, of young trees with high N2-fixing ability.