The nitrogen input through biological N2 fixation is essential for life in vast areas of the global ocean. The belief is that cyanobacteria are the only relevant N2 -fixing (diazotrophic) organisms. It has, however, now become evident that non-cyanobacterial diazotrophs, bacteria and archaea with ecologies fundamentally distinct from those of cyanobacteria, are widespread and occasionally fix N2 at significant rates. The documentation of a globally relevant nitrogen input from these diazotrophs would constitute a new paradigm for research on oceanic nitrogen cycling. Here we highlight the need for combining rate measurements and molecular analyses of field samples with cultivation studies in order to clarify the ecology of non-cyanobacteria and their contribution to marine N2 fixation on local and global scales.