Anabaena 7120 mutant 216 fails to differentiate heterocyst. We previously identified a 2.4-kb wild-type DNA fragment able to complement this mutant. We show here that the sequence of this fragment contains a single open reading frame (hetR), encoding a 299-amino-acid protein. Conjugation of deletion subclones of this fragment into strain 216 showed that the hetR-coding region is both necessary and sufficient for complementation of the Het- phenotype. The mutation in 216 is located at nucleotide 535 in the hetR gene, converting a serine at position 179 in the wild-type protein to an asparagine in the mutant. Interruption of the hetR gene in wild-type cells results in a mutant phenotype identical to that of 216. Both 216 and wild-type cells containing wild-type hetR on a plasmid display increased frequency of heterocysts, even on media containing fixed nitrogen. These results suggest that hetR encodes a product that is not only essential for but also controls heterocyst development. This putative regulatory protein lacks known structural motifs characteristic of transcription factors and probably acts at a level one or more steps removed from its target genes.