Humid tropical forests are generally characterized by the lack of nitrogen (N) limitation to net primary productivity, yet paradoxically have high potential for N loss. We conducted an intensive field experiment with 15NH4 and 15NO3 additions to highly weathered tropical forest soils in Puerto Rico to determine the relative importance of N retention and loss mechanisms. Over one-half of all the NH4+ produced was rapidly converted to NO3− via the process of gross nitrification. During the first 24 hours, plant roots took up 28% of the inorganic N produced, dominantly as NH4+, and were a greater sink for N than soil microbial biomass. Soil microbes were not a significant sink for added 15NH4+ or 15NO3− during the first 24 hours, and only for 15NH4+ after 7 days. Patterns of microbial community composition, as determined by terminal restriction fragment length polymorphism analysis (TRFLP), were weakly but significantly correlated with nitrification and denitrification to N2O. Rates of dissimilatory NO3− reduction to NH4+ (DNRA) were high in this forest, accounting for up to 25% of gross NH4+ production and 35% of gross nitrification. DNRA was a major sink for NO3−, which may have contributed to the lower rates of N2O and leaching losses. Despite considerable N conservation via DNRA and plant NH4+ uptake, the fate of ∼45% of the NO3− produced and 4% of the NH4+ produced were not measured in our fluxes, suggesting that other important pathways for N retention and loss (e.g., denitrification to N2) are important in this system. The high proportion of mineralized N that was rapidly nitrified and the fates of that NO3− highlight the key role of gross nitrification as a proximate control on N retention and loss in humid tropical forest soils. Furthermore, our results demonstrate the importance of the coupling between DNRA and plant uptake of NH4+ as a potential N-conserving mechanism within tropical forests.