The nonheme, iron-sulfur protein ferredoxin is the terminal constituent of the photosynthetic electron transport chain. Under conditions of iron stress, many cyanobacteria and eucaryotic algae replace ferredoxin with the flavoprotein flavodoxin. The gene for flavodoxin was cloned from the cyanobacterium Anacystis nidulans R2 by using three mixed oligonucleotide probes derived from the partial Synechococcus sp. strain PCC 6301 amino acid sequence. Nucleotide sequence analysis revealed a 513-base-pair open reading frame with a deduced amino acid sequence having homology to other long-chain flavodoxins. Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the A. nidulans R2 flavodoxin is 18,609. Southern blot hybridization under conditions of reduced stringency detected only one copy of the flavodoxin sequence in the A. nidulans R2 genome. Northern (RNA) blot hybridization analyses by using cloned flavodoxin gene probes indicated that no transcripts are detectable under conditions of iron saturation. However, under iron-deficient growth conditions the flavodoxin gene appeared to be transcribed as part of a larger operon. The operon yielded at least three transcripts. The first was of approximately 1,100 bases (designated RNA 1) and terminated immediately upstream from the 5' end of the flavodoxin open reading frame. A second, less abundant transcript of approximately 1,900 bases (designated RNA 2) encoded all of RNA 1 as well as the flavodoxin polypeptide. Analysis indicated that both transcripts initiate in close proximity to each other. A third, minor transcript of approximately 1,100 bases (designated RNA 3) was detectable downstream of the flavodoxin gene sequence. Addition of iron-stressed A. nidulans R2 cells resulted in almost total loss of detectable mRNA transcripts within 60 min of the addition. The ferredoxin gene transcript has previously been characterized as a monocistronic message of approximately 430 bases (M. E. Reith, D. E. Laudenbach, and N. A. Straus, J. Bacteriol. 168: 1319-1324, 1986). Here we show that the ferredoxin message is detectable under all iron regimes tested is quantitatively unaffected by decreases in iron availability to the cells.