During evolution, algae have retained a single assimilatory nitrate reductase (NR), which governs the reduction of nitrate to nitrite. The unicellular green alga Dunaliella salina is a special case, as its cells have two NRs. This makes D. salina attractive to study the mechanisms underlying the nitrate assimilation in photosynthetic unicellular organisms. Here we characterize the expression of the gene encoding the second NR, named as DsaNIA1. Low levels of mRNA for DsaNIA1 are present in ammonia-grown cells. DsaNIA1 is a nitrate- and nitrite-inducible gene. Using spectrofluorometric assays with NO-sensitive fluorescence dye, we demonstrate nitrite-dependent NO synthesis by D. salina cells. Moreover, we found that the transcription of DsaNIA1, but not DsaNIA2, is under the inducing influence of NO-dependent pathway. Together, our data argue for the two differently regulated NR isoforms in D. salina.