Abstract The ability to assimilate nitrate in non-axenic isolates of Prochlorococcus spp. was addressed in this work, particularly in three low-irradiance adapted strains originating from ocean depths with measurable nitrate concentrations. None of the studied strains was able to use nitrate as the sole nitrogen source. Nitrate reductase (NR; EC 1.6.6.2) activity was, however, detected using the methyl viologen/dithionite assay in crude extracts from all studied Prochlorococcus strains. Characterization of this activity unambiguously demonstrated its enzymatic origin. We observed that NR activity did not decrease in vivo under darkness. Attempts to detect the narB gene (coding for NR in other cyanobacteria) by PCR with primers designed on the basis of the specific codon usage in Prochlorococcus were unsuccessful. However, when primers were designed considering the codon frequencies typical of other bacteria, we could amplify different fragments of nas genes, coding for bacterial assimilatory NRs. Similar amplification products were obtained using colonies of contaminant bacteria from Prochlorococcus cultures as PCR template. Furthermore, NR activity was found in cultures of these contaminants, demonstrating the non-cyanobacterial origin of the enzyme. These results strongly suggest that the studied strains of Prochlorococcus lack NR, in spite of inhabiting environments with nitrate as the main nitrogen source. In addition, they indicate that the nitrite produced by heterotrophic bacteria is not transferred to Prochlorococcus for growth, thus discarding a trophic nitrogen chain between heterotrophic bacteria and Prochlorococcus in the studied cultures.