The presence of L-arginine methylester (AME), L-arginine ethylester (AEE), or N-nitro-L-arginine methylester (NAME) in the growth media of Staphylococcus aureus increased the nitric oxide synthase (NOS) activity approximately 5- to 14-fold. The increase of NOS activity was confirmed by two assay methods, namely assaying the formation of L-[3H] citrulline from L-[3H] arginine and NO formation. The increase of NOS activity was most likely due to increased de novo synthesis, demonstrated by Western immunoblot analysis. The addition of methanol to the culture medium also increased the NOS activity as much as that found with the above three compounds. Evidence is presented to show that AME, AEE, or NAME gave rise to the formation of methanol in vivo by the action of intracellular esterase(s) and that methanol is subsequently involved in the induction of NOS in this bacterial system.