Primary saliva is a mixture of fluid secreted through paracellular route and transcellular route. By quantification of luminal dye-dilution in the isolated acini with confocal microscopy, the transcellular fluid secretion was estimated as 25 µl/g-min. By subtraction of the transcellular fluid secretion from the whole fluid secretion, a paracellular fluid secretion was estimated to be 60 µl/g-min (1). By measurement of the 3H-labeled dextran in saliva, the two different sized paracellular routes were proposed; one is smaller than 5 Å in radius and the other is larger than 5Å (2). In addition the paracellular fluid secretion could include a pressure-dependent and a pressure-independent components; 20 and 40 µl/g-min, respectively (3). In the present study, to assess whether the pressure-dependent paracellular transport corresponds to the wider paracellular route, the submandibular salivary gland was surgically isolated from the rat (Wistar male) under anesthesia with pentobarbitone sodium (50 mg/kg body weight, i.p.). The isolated gland was perfused arterially on the stage of confocal microscope. Using sulfo-rhodamine B in the perfusate, we observed the appearance of the fluorescent dye in the intercellular canaliculi, which can pass through the larger route. The system allowed us to obtain 30 sliced images every 2 s and gave 3D reconstructed image every 2 s (5 Live, Zeiss). When the fluid secretion was induced by alpha1-adrenergic stimulation with phenylephrine, the fluorescence of intercellular canaliculi increased dose-dependently. Combined stimulation with phenylephrine and xamoterol (beta1 agonist) increased the fluorescence more than only phenylephrine. Single stimulation with isoproterenol (1 µM) decreased the fluorescence intensity in the canaliculi, suggesting lowering fluid secretion through pressure-dependent paracellular route. The present findings lead the conclusion that we could observe entry of fluorescent dye from intercellular space to luminal space by pressure-dependent paracellular route by solvent-drag, and that the fluid secretion through pressure-independent route is still open for further studies.