Estrogen receptors are localized in mitochondria, but their functions in this organelle remain unclear. We previously found that ER alpha interacted with mitochondrial protein HADHB and affected the thiolytic cleavage activity of HADHB in beta-oxidation. It is known that ER beta binds to ER alpha. In addition, ER beta is predominately located in mitochondria. These facts led us to speculate that ER beta may also be associated with HADHB in mitochondria. In order to test this hypothesis, we performed co-immunoprecipitation and confocal microscopy analyses with human breast cancer MCF7 cells. The results demonstrated that ER beta was indeed associated and colocalized with HADHB within mitochondria. Interestingly, in contrast to the stimulatory effect of ER alpha on HADHB enzyme activity observed in the previous study, silencing of ER beta enhanced the enzyme activity of HADHB in the present study, suggesting that ER beta plays an inhibitory role in HADHB enzyme activity in the breast cancer cells. Our results imply that ER alpha and ER beta may differentially affect cellular oxidative stress through influencing the rate of beta-oxidation of fatty acids in breast cancer cells. (C) 2012 Elsevier Inc. All rights reserved.