The zonal (∼15°W–40°W along 26°N–29°N) and meridional (∼30°N–30°S along 28°W–29°W) variability of δ15N of suspended particles and zooplankton (>40 µm) was studied to assess the influence of nitrogen fixation in the isotopic budget of the tropical and subtropical Atlantic ocean. Two cruises were conducted in October–November 2007 and April–May 2008 comprising a zonal and meridional transect each. In the region between 30°N and 15°N, the concurrently measured nitrogen fixation was insufficient to explain the consistent patch of suspended particles with δ15N < 2‰ and points to a significant contribution of atmospheric deposition of light nitrogen to the isotopic budget. The equatorial region (15°N–10°S) is subject to intense nitrogen fixation, which, according to a two-end-member mixing model, may explain 40–60% of the observed δ15N in suspended particles and 3–30% in zooplankton. In the South region between 10°S and 30°S, low values (<4‰) were measured in suspended particles and zooplankton during 2008. The values of δ15N of suspended particles suggest that nitrogen fixation, which is usually low (<10 µmol N m−2 day−1), may represent 50–60% of phytoplankton nitrogen in this region. Hence, diazotrophy in the South Atlantic may be more important than previously thought.