Renal kallikrein is normally synthesized and secreted by connecting tubule cells of the renal cortex. Treatment of mice with furosemide induces an expansion of the kallikrein synthesizing tubule cell population from the cortex to the inner stripe of the outer medulla. There is no induction of kallikrein gene expression in the medulla in sodium restricted mice or in untreated controls. We used hybridization histochemistry on semi-thin plastic sections to identify the medullary segment recruited to express the renal kallikrein gene after furosemide treatment. In the outer medulla, the thick ascending limb (TAL) was the only tubule segment in which kallikrein gene expression was identified. Immunogold cytochemistry revealed that kallikrein is in granules located in the cytoplasm of medullary TAL cells from furosemide-treated mice, confirming that furosemide-induced kallikrein mRNA is translated in these cells. There was no immunoreactive kallikrein found in other tubule segments of the outer medulla, nor in TAL cells from sodium or untreated mice. Granules containing immunoreactive kallikrein were secreted from both poles of the cells into the tubule lumen from the apical surface and into the interstitial space from the basolateral surface. Secretion of kallikrein from the basolateral pole of TAL cells into the interstitial space enables kinins to be generated in close apposition to the vasculature underlying renal tubules, promoting local vasodilatation. The expansion of kallikrein synthesis and secretion to the medulla after furosemide treatment may contribute to the vasodilatation which follows furosemide-induced diuresis.