Isotopic fractionation associated with N2 fixation and NO3− uptake by plants are relevant to the accuracy of estimates of N2 fixation based on differences in the natural abundance of 15N between N2 fixing and nonfixing plants. The isotope effect on N2 fixation by soybeans (Glycine max [L] Merrill, variety Harosoy) and red clover (Trifolium pratense [L]) was determined from the difference in 15N abundance between atmospheric N2 and the total N of plants grown hydroponically with N-free nutrient solution. In soybeans the isotope effect was found to be +0.98 ± 0.18‰ (β = 0.99902). In clover the isotope effect was +1.88 ± 0.14‰ (β = 0.99812). The magnitude of these inverse isotope effects is small. However, they would lead to an underestimation of the amount of N2 fixed, since the N of atmospheric origin which finally appears in the plant is made richer in 15N by the inverse isotope effects than is atmospheric N2, and, to that degree, is attributed to soil-derived N in the calculation.