The role of estrogen in breast cancer progression and activation of prolidase activity and HIF-1α led us to study the effect of estrogen on nuclear HIF-1α expression in breast cancer estrogen-dependent MCF-7 and estrogen-independent MDA-MB-231 cells. We have found that in MCF-7 cells (expressing α and β estrogen receptor) cultured without estrogen receptor activator (phenol red, estradiol), HIF-1α was down-regulated, compared to the cells cultured with estrogen receptor activator. This effect was not observed in MDA-MB-231 cells (expressing only β estrogen receptor), suggesting that α estrogen receptor is involved in down-regulation of HIF-1α. However, in MDA-MB-231 cells (expressing high prolidase activity) cultured in the presence of prolidase substrates, Gly-Pro or Gly-HyPro, HIF-1α expression was induced in a dose-dependent manner, independently of estrogen receptor activation. In MCF-7 cells (with constitutively low prolidase activity) the effect of studied iminodipeptides on HIF-1α expression was much less pronounced but it was estrogen-dependent, showing importance of prolidase activity in mechanism of this process. The data were supported by confocal microscopy bio-imaging of HIF-1α in nucleus of MCF-7 and MDA-MB-231 cells that were cultured in the presence and absence of estrogen activator and prolidase substrates. It suggests that estrogen receptor may represent important therapeutic target in pharmacotherapy of estrogen receptor positive breast cancer, while ECM degradation enzymes, including prolidase may represent target in pharmacotherapy of estrogen receptor negative breast cancers.