SUMMARY A fluorescein-labelled double-antibody technique was used for the microscopical localization of ovine prolactin in kidney tissue of rats previously injected intravenously with 50 i.u. of the hormone at varying intervals before death. Thirty seconds after the injection, the hormone could already be visualized in the lumina of the proximal tubules. After 2 min, discrete particulate fluorescence appeared on the brush border of the epithelium of the proximal tubules. Three minutes later prolactin was clearly localized in the apical portion of the cytoplasm of these cells as well as on the brush border. Ten minutes after the injection, the hormone appeared exclusively within the cell, highest concentrations being observed in the perinuclear region. After 20 min, however, the hormone appeared evenly distributed throughout the cytoplasm. The distribution of prolactin was in all cases confined to the proximal tubular region of the nephron. The findings support the suggestion that the site of action of prolactin in the kidney is the epithelium of the proximal tubule. As prolactin was first visualized in the lumen of this portion of the nephron, it appears that the hormone gains access to these cells via the glomerular filtrate.