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Human activities have profoundly altered the global nitrogen (N) cycle. Increases in anthropogenic N have had multiple effects on the atmosphere, on terrestrial, freshwater and marine ecosystems, and even on human health. Unfortunately, methodological limitations challenge our ability to directly measure natural N inputs via biological N fixation (BNF)-the largest natural source of new N to ecosystems. This confounds efforts to quantify the extent of anthropogenic perturbation to the N cycle. To address this gap, we used a pair of indirect methods -analytical modeling and N balance-to generate independent estimates of BNF in a presumed hotspot of N fixation , a tropical rain forest site in central Rondônia in the Brazilian Amazon Basin. Our objectives were to attempt to constrain symbiotic N fixation rates in this site using indirect methods , and to assess strengths and weaknesses of this approach by looking for areas of convergence and disagreement between the estimates. This approach yielded two remarkably similar estimates of N fixation . However, when compared to a previously published bottom-up estimate, our analysis indicated much lower N inputs via symbiotic BNF in the Rondônia site than has been suggested for the tropics as a whole. This discrepancy may reflect errors associated with extrapolating bottom-up fluxes from plot-scale measures, those resulting from the indirect analyses, and/or the relatively low abundance of legumes at the Rondônia site. While indirect methods have some limitations, we suggest that until the technological challenges of directly measuring N fixation are overcome, integrated approaches that employ a combination of model-generated and empirically-derived data offer a promising way of constraining N inputs via BNF in natural ecosystems. Study Area