Dinitrogen (N2) fixation is recognized as an important input of new nitrogen (N) to the open ocean gyres, contributing to the export of organic matter from surface waters. However, very little N2-fixation research has focused on the South Atlantic Gyre, where dust deposition of iron (Fe), an important micronutrient for diazotrophs, is seasonally low. Recent modeling efforts suggest that N2-fixation may in fact be closely coupled to, and greatest in, areas of denitrification, as opposed to the oceanic gyres. One of these areas, the Benguela Upwelling System, lies to the east of the South Atlantic Gyre. In this study we show that N2-fixation in surface waters across the South Atlantic Gyre was low overall (<1.5 nmol N l?1 d?1) with highest rates seen in or near the Benguela Upwelling System (up to ?8 nmol N l?1 d?1). Surface water dissolved Fe (dFe) concentrations were very low in the gyre (?0.3 nM or lower), while soluble reactive phosphorus (SRP) concentrations were relatively high (?0.15 ?M). N2-fixation rates across the entire sampling area were significantly positively correlated to dFe, but also to SRP and NO3?. Thus, high NO3? concentrations did not exclude N2-fixation in the upwelling region, which provides evidence that N2-fixation may be occurring in previously unrecognized waters, specifically near denitrification zones. However the gene encoding for a nitrogenase component (nifH) was not detected from known diazotrophs at some stations in or near the upwelling where N2-fixation was greatest, suggesting the presence of unknown diazotrophs in these waters.