The flavohaemoglobin gene, hmp, of Escherichia coli is upregulated by nitric oxide (NO) in a SoxRS-independent manner. We now show that hmp expression is also upregulated by S-nitrosoglutathione (GSNO, widely used as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ donor). Elevated homocysteine (Hcy) levels, achieved either by adding Hcy extracellularly or using metE mutants, decreased hmp expression. Conversely, metC mutants (defective in Hcy synthesis) had higher levels of hmp expression. Mutations in metR abolished hmp induction by GSNO and SNP, and hmp expression became insensitive to Hcy. We propose that the previously documented modulation by Hcy of MetR binding to the glyA-hmp intergenic regulatory region regulates hmp transcription. Although two MetR binding sites are present in this region, only the higher affinity site proximal to hmp is required for hmp induction by GSNO and SNP. GSNO and SNP react with Hcy in vitro under physiologically relevant conditions of pH and temperature generating S-nitrosohomocysteine, although in the latter case this would be co-ordinated to the Fe in SNP as a stable species. The free S-nitrosocysteine generated in the reaction with GSNO breaks down to release NO more readily than via homolysis of GSNO. As GSNO and SNP upregulate hmp similarly, the NO released in the former case on reaction with homocysteine cannot be involved in hmp regulation.