A Nitric-oxide synthase-like (NOS) protein has been found to be present in several Gram-positive bacteria, including Bacillus subtilis. NOS generates nitric-oxide from the amino acid L-arginine via the stable intermediate N-hydroxy-L-arginine. The function of NO production in Gram-positive bacteria has not yet been elucidated, but studies indicate a function in signal transduction. In this study, a proteomic approach is used to examine the physiological role of NO in the Gram-positive model bacteria, B. subtilis. Protein profiles obtained from two-dimensional gel electrophoresis of cells grown in the presence of aminoguanidine, a known NOS inhibitor, revealed the down-regulation of three proteins via NO inhibition over a pI range of 4–7 when compared to an uninhibited sample. These three proteins were identified by LC-MS/MS as a nucleoside diphosphate kinase, an MreB-like protein, and a phage shock A protein homolog. The down-regulation of these proteins via NOS inhibition provides preliminary evidence that NO plays a signal transduction role in Gram-positive bacteria.