Nitric oxide synthases (NOS) are monooxygenase enzymes that catalyze the oxidation of L-arginine to L-citrulline and nitric oxide (NO). They are composed of a catalytic heme binding domain (NOSox) and a flavin-binding domain (NOSred) responsible for electron transfer and heme activation. NOS-derived NO serves as a signaling molecule in animals, controlling vascular tone, immune response, and neuronal signaling. NOS are also found in bacteria and are involved in various roles, including biofilm formation, recovery from UV damage, and protection from oxidative stress. However bacterial NOS sequences only contain a NOSox domain, and must rely on nonspecific reductases for activation. Recently, sequences for a unique NOS-like protein have been identified in cyanobacteria. These proteins are the first bacterial NOS to contain a mammalian-like NOSred, and also contain a globin-like domain (NOSg) which has not been observed in any other NOS. This work confirms that the NOS from the cyanobacteria Synechoccocus sp. PCC 7335 (syNOS) is a true NOS, and produces NO from L-arginine. However, the factors governing syNOS activation deviate from our current knowledge of NOS enzymology. syNOS requires Ca2+ and tetrahydrobiopterin for NO production, and syNOSg rapidly oxidizes all NO to nitrate. syNOSred facilitates the reduction of syNOSox and syNOSg independent of one another, which indicates direct electron transfer between syNOSg and syNOSred. The reduction of syNOSg can also be mediated by syNOSFAD, in a manner analogous to flavohemoglobin proteins, and does not require syNOSFMN, as in NOSox reduction. The structures of syNOSFAD and syNOSFMN have been determined, and homology modeling of syNOSg confirms that syNOSFAD – syNOSg domain interactions are possible. The function of syNOS has yet to be identified, but genomic analysis of other syNOS homologues suggests it may participate in signal transduction pathways. The presence of syNOSg and its Ca2+ dependence may serve as a switch to turn on/off such signaling pathways.