Cathepsin D (CD), a lysosomal aspartic protease present in mammary tissue and milk in various molecular forms, is also found in the incubation medium of mammary acini in molecular forms that are proteolytically active on prolactin at a physiological pH. Because prolactin controls the vesicular traffic in mammary cells, we studied, in vivo and in vitro, its effects on the polarised transport and secretion of various forms of CD in the rat mammary gland. CD accumulated in vesicles not involved in endocytosis, in the basal region of cells. Prolactin increased this accumulation and the release of endosomal active single chain CD at the basal side of acini. The CD-mediated proteolysis of prolactin, leading to the antiangiogenic 16kDa form, at a physiological pH, was only observed in conditioned medium but not in milk. These data support the novel concept that an active molecular form of CD, secreted at the basal side of the mammary epithelium, participates in processing blood-borne prolactin outside the cell, this polarised secretion being controlled by prolactin itself.