There is increasing interest in the bidirectional communication between the mammalian host and prokaryotic cells. Catecholamines (CA), candidate molecules for such communication, are presumed to play an important role in the gut lumen; however, available evidence is limited because of the lack of actual data about luminal CA. This study evaluated luminal CA levels in the gastrointestinal tract and elucidated the involvement of gut microbiota in the generation of luminal CA by comparing the findings among specific pathogen-free mice (SPF-M), germ-free mice (GF-M), and gnotobiotic mice. Substantial levels of free dopamine and norepinephrine were identified in the gut lumen of SPF-M. The free CA levels in the gut lumen were lower in GF-M than in SPF-M. The majority of CA was a biologically active, free form in SPF-M, whereas it was a biologically inactive, conjugated form in GF-M. The association of GF-M with either Clostridium species or SPF fecal flora, both of which have abundant β-glucuronidase activity, resulted in the drastic elevation of free CA. The inoculation of E. coli strain into GF-M induced a substantial amount of free CA, but the inoculation of its mutant strain deficient in the β-glucuronidase gene did not. The intraluminal administration of DA increased colonic water absorption in an in vivo ligated loop model of SPF-M, thus suggesting that luminal DA plays a role as a proabsorptive modulator of water transport in the colon. These results indicate that gut microbiota play a critical role in the generation of free CA in the gut lumen.