Intralobular distribution of Ito cells (fat-storing cells) in hepatic microcirculatory units was investigated through intravital fluorescence microscopy. By using a low-level ultraviolet epi-illumination and a sensitive silicon intensified target camera, the intrahepatic autofluorescence was visualized and digitally processed. In rats fed an ordinary diet, the ultraviolet-excited autofluorescence was composed of at least two different origins that could not be spectrophotometrically distinguished, namely, multiple patchy autofluorescence activities along the sinusoids and terminal hepatic venules and diffuse parenchymal autofluorescence. Multiple patchy activities showed a rapid photobleaching phenomenon under the continuous ultraviolet excitation. These fluorescent activities were completely eliminated by depleting the intrahepatic retinoid contents using a vitamin A-deficient diet for 4 weeks. Furthermore repeated administration of vitamin A significantly enhanced the patchy fluorescent activities. Electron microscopy revealed that these vitamin A fluorescent activities are colocalized with the fat droplets in Ito cells, providing evidence that Ito cells exist not only in perisinusoidal spaces but also in the perivascular spaces of the terminal hepatic and portal venules. The current intravital technique thus provides a new method to observe Ito cells in hepatic microcirculatory units in vivo.