We evaluated the morphology and physiologic function of the bile canaliculi (BC) in the rat liver slice (RLS) by confocal laser scanning microscopy (CLSM). Lucifer yellow (LY) dye was injected into the RLS, and the distribution of LY was serially evaluated. After the injection of LY, hepatocytes were initially visualized, followed by visualization of the BC. There was no significant difference in the distribution of LY between zones 1 and 3 in the hepatic lobule. In zone 1, the reticular distribution of the BC was observed, whereas the part of BC was linearly visualized in zone 3 along the course of sinusoids. When changes in the bile canalicular fluorescence (BCF) were serially evaluated, the BCF was decreased to the minimal level (88% of the value obtained immediately after the LY injection) 10 min after the LY injection, and it tended to increase thereafter. The intralobular hepatocyte fluorescence (ILHF) was decreased to 58.9% of the initial value during the first 40 min. However, the ILHF was transiently increased 30 min after the LY injection, suggesting the possibility of reabsorption of LY by hepatocytes. Three-dimensional (3-D) reconstruction images of the BC facilitated the evaluation of the stereoscopic structure of BC. Confocal laser scanning microscopy facilitated the evaluation of structures and physiologic function of the BC.