Mixtures of Eucalyptus globulus Labill. and Acacia mearnsii de Wildeman are twice as productive as E. globulus monocultures growing on the same site in East Gippsland, Victoria, Australia, possibly because of increased nitrogen (N) availability owing to N(2) fixation by A. mearnsii. To investigate whether N(2) fixation by A. mearnsii could account for the mixed-species growth responses, we assessed N(2) fixation by the accretion method and the (15)N natural abundance method. Nitrogen gained by E. globulus and A. mearnsii mixtures and monocultures was calculated by the accretion method with plant and soil samples collected 10 years after plantation establishment. Nitrogen in biomass and soil confirmed that A. mearnsii influenced N dynamics. Assuming that the differences in soil, forest floor litter and biomass N of plots containing A. mearnsii compared with E. globulus monocultures were due to N(2) fixation, the 10-year annual mean rates of N(2) fixation were 38 and 86 kg ha(-1) year(-1) in 1:1 mixtures and A. mearnsii monocultures, respectively. Nitrogen fixation by A. mearnsii could not be quantified on the basis of the natural abundance of (15)N because such factors as mycorrhization type and fractionation of N isotopes during N cycling within the plant confounded the effect of the N source on the N isotopic signature of plants. This study shows that A. mearnsii fixed significant quantities of N(2) when mixed with E. globulus. A decline in delta(15)N values of E. globulus and A. mearnsii with time, from 2 to 10 years, is further evidence that N(2) was fixed and cycled through the stands. The increased aboveground biomass production of E. globulus trees in mixtures when compared with monocultures can be attributed to increases in N availability.