Charcoal-stripped fetal bovine serum (CS-FBS) is frequently used in studies on hormone-responsive cancers to provide hormone-free cell culture conditions. CS-FBS may influence the growth of cancer cells; however, the underlying mechanisms remain unclear. In this study, we aimed to clarify the effects of CS-FBS on distinct subtypes of breast cancer cells. We found that the crucial oncoprotein c-Myc was significantly inhibited in estrogen receptor alpha (ER-alpha)-positive breast cancer cells when cultured in CS-FBS-supplemented medium, but it was not suppressed in ER-alpha-negative cells. The addition of 17 beta-estradiol (E2) to CS-FBS-supplemented medium rescued the CS-FBS-induced inhibition of c-Myc, while treatment with 5 alpha-dihydrotestosterone (DHT) suppressed c-Myc expression. Our data demonstrated that CS-FBS may impede the growth of ER-alpha-positive breast cancer cells via c-Myc inhibition, and this was possibly due to the removal of estrogen. These results highlighted that the core drivers of c-Myc expression were subtype-specific depending on the distinct cell context and special caution should be exercised when using CS-FBS in studies of hormone-responsive cancer cells. (C) 2020 Elsevier Inc. All rights reserved.