Mammary epithelial cells (MEC) of lactating animals ferry large amounts of milk constituents in vesicular structures which have mostly been characterized by morphological approaches (Ollivier-Bousquet, 1998). Recently, we have shown that under conditions of lipid deprivation, perturbed prolactin traffic paralleled changes in the membrane phospholipid composition and in the cytosol versus membrane distribution of annexin VI (Ollivier-Bousquet et al., 1997). To obtain additional information on the membrane events involved in the vesicular transport of the hormone to the apical pole of the cell, we conducted a biochemical study on prolactin-containing vesicles in MEC at two different stages of differentiation. We first showed that MEC of pregnant and lactating rabbits exhibited membrane characteristics of non-polarized and polarized cells respectively, using annexin IV and the alpha-6 subunit of integrin as membrane markers. Incubation of both cell types with biotinylated prolactin for 1 h at 15 degrees C, followed by a 10-min chase at 37 degrees C revealed that prolactin transport was activated upon MEC membrane polarization. This was confirmed by subcellular fractionation of prolactin-containing vesicles on discontinuous density gradients. In non-polarized MEC, (125)I-prolactin was mainly recovered in gradient fractions enriched with endocytotic vesicles either after incubation at 15 degrees C or after a 10-min chase at 37 degrees C. In contrast, in polarized MEC, the hormone switched from endocytotic compartments to a fraction enriched in exocytotic clathrin-coated vesicles during the 10-min chase at 37 degrees C. Association of annexin VI to prolactin carriers was next studied in both non-polarized and polarized cells. Membrane compartments collected at each gradient interface were solubilized under mild conditions by Triton X-100 (TX100) and the distribution of annexin VI in TX100-insoluble and TX100-soluble fractions was analyzed by Western blotting. Upon MEC polarization, the amount of annexin VI recovered in TX100-insoluble fractions changed. Quite interestingly, it increased in a membrane fraction enriched with endocytotic clathrin-coated vesicles, suggesting that annexin VI may act as a sorting signal in prolactin transport.