Probiotic bacteria elicit a number of beneficial effects in the gut but the mechanisms for these health promoting effects are not entirely understood. Recent in vitro data suggest that lactobacilli can utilise nitrate and nitrite to generate nitric oxide, a gas with immunomodulating and antibacterial properties. Here we further characterised intestinal NO generation by bacteria. In rats, dietary supplementation with lactobacilli and nitrate resulted in a 3-8 fold NO increase in the small intestine and caecum, but not in colon. Caecal NO levels correlated to nitrite concentration in luminal contents. In neonates, colonic NO levels correlated to the nitrite content of breast milk and faeces. Lactobacilli and bifidobacteria isolated from the stools of two neonates, generated NO from nitrite in vitro, whereas S. aureus and E. coli rapidly consumed NO. We here show that commensal bacteria can be a significant source of NO in the gut in addition to the mucosal NO production. Intestinal NO generation can be stimulated by dietary supplementation with substrate and lactobacilli. The generation of NO by some probiotic bacteria can be counteracted by rapid NO consumption by other strains. Future studies will clarify the biological role of the bacteria-derived intestinal NO in health and disease.