Nitrogenase activity, indicative of N2 fixation, was measured in the surface waters along a north–south transect in the eastern Atlantic Ocean, from Texel (The Netherlands, 53°N) to Cape Town (South Africa, 35°S) using a sensitive on-line, near real-time acetylene reduction assay. From the beginning of January to the end of February 2000 nitrogenase activity was detected in varying rates, but only between 14°N and 13°S latitudes. Dark incubations yielded an average activity of 2.2 (± 2.4) µmol m-2 d-1 N, but light increased the activity to 3.7 (± 2.9) µmol m-2 d-1 N. However, nitrogenase activity in the light was sensitive to O2 doubling to 7.6 (± 12.7) µmol m-2 d-1 N when the incubation was anaerobic. In the area where N2 fixation occurred, phosphate concentrations were fourfold lower than in the area where N2 fixation was absent, while silicate levels were higher. The water temperature in the area with N2 fixation was 28°C, while in the adjacent area the temperature was 3°C lower, which might have prevented the proliferation of diazotrophic cyanobacteria. Action spectra revealed that chlorophyll a, phycocyanin, and phycoerythrin are the light-harvesting pigments supporting nitrogenase activity. In one area in the northern latitudes, potential nitrogenase activity was highest during daytime, which is characteristic for Trichodesmium. In areas with a high potential nitrogenase activity, surface waters were dominated by a phycoerythrin-containing cyanobacterium. Since nitrogenase activities were highest at night, these cells may have been unicellular cyanobacteria like Crocosphaera.