Quantitative estimates of biological N-2 fixation (BNF) in Robinia pseudoacacia, one of the few temperate N-2-fixing trees (NFTs) are lacking. A field experiment was conducted to measure N-2 fixation in R. psedoacacia over a 2-year period, using the N-15 isotope dilution (ID) method. Two non-NFTs, Ligustrum vulgaris and Syringa vulgare, served as reference plants. In general, errors occurred in using the ID method to measure N-2 fixation when the dilution of N-15 by the unlabelled N in plants at transplanting was not taken into account. This could have serious implications for estimating N-2 fixation in trees in established stands. For the first year, dissimilar values of N-2 fixation were measured by the two reference plants. However, for the second year, or for the entire 2 years, similar values were obtained. From this study, we deduced that R. pseudoacacia has a high potential for N-2 fixation. At the end of the first year, the percentage of N derived from fixation (% Ndfa) averaged for the two reference trees was 65%, and 90% in the second year. The overall % Ndfa for the 2-year period was 80. The equivalent amounts fixed were, 12.2 and 25.8 g N tree(-1) in the first and second years, respectively. At the planting rate used, 5714 plants ha(-1), this would be equivalent to about 220 kg N ha(-1) within the 2 years. Although all the aboveground parts together gave % Ndfa values close to that for the whole tree, none of the individual plant parts gave an accurate estimate of total N fixed in R. pseudoacacia, and excluding the N in roots resulted in underestimates of total N fixed by about 14.7, 37.5 and 30.3% in the first, second year and the total for the entire 2 years, respectively. Thus, for N balance studies in ecosystems, it is necessary to destructively sample all plant parts in order to obtain accurate estimates of the contribution from BNF.