ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N 2 in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.