The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. We have recently reported the design, synthesis, and preliminary evaluation of a doxorubicin-formaldehyde conjugate targeted, via 4-hydroxytamoxifen, to the estrogen receptor (ER) and antiestrogen binding site (AEBS), which are commonly present in breast cancer cells. The lead targeted doxorubicin-formaldehyde conjugate, called DOX-TEG-TAM, was found to possess superior cell growth inhibition characteristics relative to clinical doxorubicin and an untargeted control conjugate, especially in ER-negative, multidrug resistant MCF-7/Adr cells. The enhanced activity in the absence of estrogen receptor raised the possibility that targeting was also mediated via AEBS. Fluorescence microscopy of an ER-negative, AEBS-positive cell line as a function of time showed initial DOX-TEG-TAM localization in cytosol, in contrast to initial DOX and untargeted doxorubicin-formaldehyde conjugate localization in the nucleus. DOX-TEG-TAM was taken up by four AEBS-positive cell lines to a greater extent than doxorubicin and an untargeted doxorubicin-formaldehyde conjugate. Of the four cell lines, three were ER negative. DOX-TEG-TAM uptake was inhibited in a dose-dependent manner by the presence of a competing AEBS ligand. DOX-TEG-TAM retains 60% of the affinity of 4-hydroxytamoxifen for AEBS. DOX-TEG-TAM was also taken up by the AEBS-negative, ER-positive cancer cell line Rtx-6; with these cells uptake was inhibited in a dose-dependent manner by the ER ligand, estradiol. The data support the hypothesis that uptake of 4-hydroxytamoxifen targeted doxorubicin-formaldehyde conjugate is mediated by both the antiestrogen binding site and estrogen receptor.