We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633 nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labelled insulin was injected twice (the second time after ~140 min) into the right jugular vein and the fluorescence signal (at 650-670 nm) recorded. Fluorescence was detected almost immediately at the brush border (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio 2.11±0.26, n=10; mean±SEM), indicating a 'priming', or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pre-treatment with intravenous lysine (ratio 1.03±0.07, n=6; P<0.01). Cy2 or Cy3 labeled insulin uptake in a PT cell line in vitro was monitored by 488 nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semi-quantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (up-regulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand 2-glycoprotein.