Drug resistance occurs frequently during breast cancer treatment with antiestrogens. Since antiestrogen action is mediated by the estrogen receptor (ER), we have examined both the structural and functional properties of the ER present in two breast cancer cell lines, LY2 and T47D, which proliferate rapidly in the presence of antiestrogens. The ER function in LY2 cells was indistinguishable from that of the parental tamoxifen-sensitive MCF-7 cells as assessed by estrogen regulation of two endogenous genes and estrogen-regulated transcription in a transient transfection system. RNase protection assays, sensitive enough to detect single base pair mismatches, showed that the sequence of the coding region of ER of LY2 and T47D cells was wild type. Thus the ER appears to be normal in two independently isolated breast cancer cell lines whose growth is resistant to the inhibitory effect of antiestrogens. Moreover by conducting the cell proliferation studies in a phenol red-free medium, we have demonstrated that the antiestrogen resistance of LY2 and T47D cells corresponds in fact to an estrogen-independent growth.