Nitric oxide (NO) is an important signal and effector molecule. It is produced from the amino acid L-arginine by a family of enzymes known as NO synthases. Although NO biosynthesis through denitrification has been well studied in microorganisms, the formation of NO from L-arginine has rarely been investigated. The NO, produced by commensal bacteria, may influence greatly the metabolism of mammals. Lactobacilli represent components of the commensal mammalian gastrointestinal microbiota. The functional importance of Lactobacillus species in the mammalian intestine highlights the need for detailed studies of the occurence of NO synthesis in this bacteria. Three strains of L. fermentum and L. plantarum were investigated for nitric oxide production. NO-synthase activity has been demonstrated for L. plantarum N8P-A3 and L. fermentum N90T-C4 using EPR. The possibility of nonenzymatic NO generation was also demonstrated. None of the strains of L fermentum and L plantarum examined had denitrifying nitrite reductase activity under anaerobic conditions. Furthermore, NO in L. plantarum N52 and L. fermentum AL1 has been directly visualized using the NO-specific fluorescent probes DAF-FM DA and DAA. The spatial spread of NO in bacterial cells was revealed. Thus, Lactobacillus produce NO through nitric oxide synthase system.