This study investigated the bulk nitrogen isotopic composition and fractionation of marine and freshwater cyanobacteria growing at the expense of atmospheric dinitrogen gas (N2) or nitrate (NO3-). In agreement with previous studies, cyanobacteria using N2 had d15N values ranging from -2‰ to -1‰ and fractionated by 2–3‰ against atmospheric N2. When grown on NO3-, the analyzed cyanobacteria had a remarkably large range in d15N, from -14‰ to 1‰, and fractionated by 4–19‰ relative to the supplied NO3-. Interestingly, several of the NO3--utilizing cyanobacteria expressed a nitrogen isotopic signal similar to the one expected for diazotrophic species. Extrapolation to the natural environment indicates that d15N signals considered to be associated with dinitrogen fixation could also be generated by NO3- utilization, depending on the isotopic composition of the nitrogen source, the degree of fractionation, and the type of cyanobacterium involved.