The 15N-enrichment technique for estimating biological nitrogen fixation in Casuarinaequisetifolia J.R. & G. Forst. was evaluated under field conditions in single-species and mixed-species plantings (with a nonfixing reference species, Eucalyptus × robusta J.E. Smith) between ages 6 and 24 months in Puerto Rico. Trenched and untrenched quadrats within the plantations were labelled at 6-month intervals with 15N-enriched ammonium sulfate at a rate of 0.2 g 15N•m−2•year−1 (2.0 g N•m−2•year−1). Analyses of foliar and whole-tree (weighted average) N-isotope ratios, based on periodic nondestructive and whole tree harvests, were used to estimate the proportion of nitrogen derived from biological dinitrogen fixation (PNDFA) and total nitrogen derived from fixation (TNDFA) in Casuarina. These results were compared with those obtained using the nitrogen difference method. The 15N-enrichment technique yielded consistent estimates of biological nitrogen fixation in Casuarina when either foliar or whole-tree N-isotopic data were used to calculate PNDFA and TNDFA. Estimates of PNDFA in Casuarina were similar for trenched monoculture and mixed-species quadrats, and in trenched and untrenched quadrats where the reference species (Eucalyptus) was interplanted with the N-fixing species. However, the results indicate that eucalyptus grown in small, untrenched monoculture quadrats is an inappropriate reference for estimating PNDFA in Casuarina. During the first 2 years after plantation establishment, Casuarina obtained from 48 to 67% of its nitrogen from the atmosphere based on foliar and whole-tree sampling. This amounted to between 82 and 94 kg•ha−1•year−1 in the monoculture treatment and between 39 and 62 kg•ha−1•year−1 in the mixed stands with Eucalyptus. These results also were in close agreement with estimates made using an N-difference method.