Cathepsin-D and pS2 are two estrogen-regulated proteins in human breast cancer cell lines. They have been considered possible prognostic factors in breast cancer, but results have been contradictory. To better understand the regulation of these proteins, we investigated the role of estradiol (E2), serum, and growth factors in hormone-dependent (MCF-7, ZR75.1) and hormone-independent (MDAMB-231, BT20) breast cancer cell lines. E2 treatment in serum-free conditions increased intracellular and secreted levels of pS2 in ZR75.1 and in MCF-7, secreted levels only of cathepsin-D in MCF-7, and both levels of cathepsin-D in ZR75.1. Insulin-like growth factor I (IGF-I) and progesterone receptors were also stimulated by E2, whereas the estrogen receptor was down-regulated. Following treatment with epidermal growth factor (EGF), secreted pS2 levels doubled only in MCF-7 cells. IGF-I did not modify cathepsin-D or pS2 levels in either cell line, but caused an increase in its own receptor. Cathepsin-D and pS2 doubled in MCF-7 cells grown in medium supplemented with denaturated serum, but estrogen regulation of these proteins was still maintained. Cathepsin-D was expressed in MDAMB-231 and BT20, but its levels were modified by neither E2 nor growth factor treatment. Conversely, neither cell line expressed detectable levels of pS2 before or after treatment. In conclusion, our results show that in different types of breast cancer cells, some estrogen-regulated proteins (e.g. pS2) are also regulated by growth factors-such as EGF and other unknown serum factors. This may account for the contradictory results obtained regarding the prognostic relevance of cathepsin-D and pS2.