The reported estrogenic action of phenol red and/or its lipophilic contaminants has led to the widespread use of indicator-free culture medium to conduct endocrine studies in vitro. Because we have recently developed methods to measure large-magnitude estrogen effects in the tissue culture medium containing phenol red, we concluded that the indicator issue required further evaluation. To do this, we selected nine estrogen receptor positive (ER ) cell lines representing four target tissues and three species. We investigated phenol red using five different experimental protocols. First, 17β-estradiol (E₂) responsive growth of all nine ER cells lines was compared in the medium with and without the indicator. Second, using representative lines we asked if phenol red was mitogenic in the indicator-free medium. The dose–response effects of phenol red were compared directly to those of E₂. Third, we asked if tamoxifen-inhibited growth equally in phenol red–containing and indicator-free medium. This study was based on a report indicating that antiestrogen effects should be seen only in phenol red–containing medium. Fourth, we asked if phenol red displaced the binding of ³H-E₂ using ER intact human breast cancer cells. Fifth, we compared E₂ and phenol red as inducers of the progesterone receptor using a human breast cancer cell line. All the experiments presented in this report support the conclusion that the concentration of phenol red contaminants in a standard culture medium available today is not sufficient to cause estrogenic effects. In brief, our studies indicate that the real issue of how to demonstrate estrogenic effects in culture resides elsewhere than phenol red. We have found that the demonstration of sex steroid hormone–mitogenic effects in culture depends upon conditions that maximize the effects of a serum-borne inhibitor(s). When the effects of the inhibitor are optimized, the presence or absence of phenol red makes no everyday difference to the demonstration of estrogen mitogenic effects with several target cell types from diverse species.