Nitric oxide (NO) is a central mediator of various physiological events in the gastro-intestinal tract. The influence of the intestinal micro-flora for NO production in the gut is unknown. Bacteria could contribute to this production either by stimulating the mucosa to produce NO, or they could generate NO themselves. Using germ-free and conventional rats we measured gaseous NO directly in the gastro-intestinal tract and from the luminal contents using a chemiluminescence technique. Mucosal NO production was studied using a NO synthase inhibitor and to evaluate microbial contribution to the NO generation, nitrate was given to the animals. In conventional rats, luminal NO differed profoundly along the gastro-intestinal tract with greatest concentrations in the stomach (> 4000 ppb), caecum (~ 200 ppb) and lower in the small intestine and colon ( 20 ppb). Caecal NO correlated to the levels in incubated luminal contents. NO synthase inhibition lowered NO levels in the colon, without affecting NO in the stomach and in the caecum. Gastric NO increased greatly after a nitrate load, proving it to be a substrate for NO generation. In germ-free rats, NO was low ( 30 ppb) throughout the gastro-intestinal tract and absent in the incubated luminal contents. NO also remained low also after a nitrate load. Our results demonstrate a pivotal role of the intestinal micro-flora in gastro-intestinal NO generation. Distinctly compartmentalised qualitative and quantitative NO levels in Conv and GF rats reflects complex host-microbial cross-talks, possibly making NO a regulator of the intestinal eco system.