In vivo fluorescent microscopy with direct observation of flow through the islet was used to investigate the islet microcirculation. In urethane-anesthetized rats (n= 18), the pancreas was exposed and an islet was identified under direct microscopy. The vertical illuminator for fluorescent microscopy was turned on and fluorescein-albumin conjugate or fluorescent microspheres were injected intravenously or intraarterially. Each study was videotaped; on slow motion playback, the flow of the conjugate or microspheres was followed through the islet, the islet capillaries, and then to venules exiting the islet. One islet in the head of the pancreas in 12 rats was studied. The arterioles first reached the surrounding mantle of the islet where they divided into capillaries that carried conjugate or microspheres to other portions of the mantle or the core of the islet. Flow of conjugate traversed the core and returned to different portions of the mantle. The fluorescent microsphere study permitted a more detailed study of the pathways followed, the individual microspheres being seen to travel through numerous tortuous pathways through the islet. The flow of microspheres was nonhomogeneous in that individual microspheres in one portion of the islet would stop, then move on, while other microspheres flowed freely. The capillaries joined two to six venules that carried the conjugate or microspheres out of the islet. One or two of the exiting microvessels entered the adjacent acinar microcirculation; the others entered larger collecting venules. In six tail islets studied, the microcirculation was similar to that of the islets in the head of the pancreas. The fluorescent in vivo microscopy technique permitted delineation of a complex pattern of flow through the islet microvasculature that suggests there is not a simple B-to A to D-cell order of cellular perfusion.