Nitric oxide (NO) is a key regulator of diverse biological processes, including the modulation of blood vessel tone [1]. Nitric oxide synthase (NOS), which oxidizes arginine to produce NO and citrulline [2], is found in organisms from bacteria to humans. Despite the impact of NO on physiology, mice lacking all three mammalian NOS isoforms develop to term and are viable [3]. There is a single NOS ortholog encoded in the Drosophila genome (Nos). Regulski et al. [4] described a mutation in a conserved residue that abrogates NOS activity, and reported that this lesion confers lethality (Nos C ). However, two lines of evidence led us to believe that this lethality could be due to a closely associated mutation rather than the lesion in Nos itself. First, the lethality was not rescued by reintroduction of NOS. Second, while the authors convincingly demonstrate that they have generated a mutation in the Nos gene that inactivates the enzyme, they do so for only one of the 17 alleles that they assign to the Nos complementation group. Beginning with a stock of Nos C provided by Regulski et al. [4], we isolated recombinant chromosomes in which we separated the lethal lesion from the point mutation in Nos C . Additionally, we generated a deletion that removes significant portions of the Nos coding sequences, including those responsible for synthesis of NO, and found it to be homozygous viable. Both our deletion and Nos C eliminate NOS enzymatic activity without affecting Drosophila development, and without obviously compromising the health of the flies.