NO2- effux and its regulation have been studied in the cyanobacterium Nostoc MAC. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), sodium azide, p-chloromercuribenzoate (PCMB), and dicyclohexylcarbodiimide (DCCD), a specific inhibitor of bacterial ATPase, inhibited the NO2- efflux activity significantly. No NO2- efflux activity was observed under dark-aerobic as well as under dark-anaerobic conditions; however, the addition of ATP resulted in NO2- efflux even under dark-aerobic condition. Maximum NO2- efflux activity was observed when NO3- served as the sole nitrogen source. However, NH4+ ions inhibited the NO2-efflux activity when both NO3- and NH4+ were simultaneously available to the cells. The NO2- efflux was freed from NH4+ repression by L-methionine-DL-sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Chloramphenicol, a protein synthesis inhibitor, inhibited the derepression of NO2- efflux system when NH4+-incubated cells were transferred to NO3- medium. Tungstate-treated cells lacking functional NO3- reductase but having NO3- uptake activity also lacked NO2- efflux activity. These results suggest that (i) NO2-efflux in Nostoc MAC is NO3- dependent and an energy-dependent process that can be regulated at the levels of NO3- uptake and NO3- reductase; (ii) NO2- efflux system is NH4+ repressible; however, the product of NH4+ assimilation via GS is being required for repression to occur; (iii) de novo protein synthesis is required for derepression of the NO2- efflux system; and (iv) the catalytic activity of NO2- reductase also seems to play an important role in the regulation of NO2- efflux system.