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Physiological roles of endogenous nitric oxide (NO) in the lymphatic pump activity of rat mesenteries in vivo were evaluated using an intravital video microscope system. Changes in the pumping frequency ( F), the end diastolic diameter (EDD), and the end systolic diameter (ESD) of the mesenteric lymph microvessels were measured with the microscope system and then the pump flow index (PFI) was calculated. A 15-min superfusion of 30 μM N ω-nitro-l-arginine methyl ester (l-NAME) in the mesenteries caused significant increases of F and PFI and a significant decrease of the EDD and ESD. Simultaneous superfusion of 1 mM l-arginine with 30 μMl-NAME produced a significant reversal of thel-NAME-mediated increase of F and decrease of ESD. A 15-min superfusion of 100 μM aminoguanidine caused no significant effects on F, EDD, and ESD of the mesenteric lymph vessels in vivo. These findings suggest that endogenous NO has physiologically modulated the lymphatic pump activity in rat mesentery in vivo and that the production and release of NO may be mediated by constitutive NO synthase but not by inducible NO synthase.