Human activities have greatly increased biologically available nitrogen (N) worldwide, with rapidly increasing rates of deposition in tropical regions. Nitrogen deposition is likely to alter N cycling in natural ecosystems, including inputs of N via biological N fixation (BNF). We determined rates of BNF in a lower montane and a montane tropical forest in Puerto Rico, and assessed the impact of N additions on rates of BNF using an on-going N fertilization experiment. Rates of BNF were measured in the forest soil, litter layer, and canopy epiphylls, lichens and moss using acetylene reduction assays. The highest background rates of BNF were found in the litter layer for the lower montane forest (1.3 ± 0.3 kg N/ha/y, mean ± s.e.), and in the canopy for the montane forest (1.7 ± 0.1 kg N/ha/y). Total background BNF was 3.1 ± 0.1 kg N/ha/y in the lower montane forest, and 3.3 ± 0.1 kg N/ha/y in the montane forest. Nitrogen additions significantly increased N concentrations in the forest floor for both forest types, and in canopy leaves for some species in the montane forest. Nitrogen fertilization also decreased rates of forest floor BNF by a factor of 10 in both forest types. For the montane forest canopy, tree species had a significant effect on BNF rates, as did fertilization (p < 0.05). We conclude that N fixation is an important input of N to both of these systems, and that N deposition in tropical regions has the potential to suppress BNF in various forest compartments.