Tamoxifen is the widespread anti-hormonal compound used for the treatment of human breast cancer. It is admitted that its effects are mediated via estrogen receptors (ER) but the molecular basis of its activity has yet to be clearly defined. In this work, we have developed a new image cytometry procedure in order to clarify the interactions between steroid receptors and tamoxifen at the cell cycle kinetic level. On untreated cells, an increase of ER level and a decrease of progesterone receptor (PR) level during the G0/G1 phase were demonstrated. Then, the ER and PR levels fell during the S-phase until the beginning of G2/M phase, where an increase was observed, especially for PR. These results suggest that ER is synthesized preferentially during the G0/G1 transition and PR during the S/G2 transition. After short-term tamoxifen treatment an augmentation of ER level was observed which was not dose-dependent, suggesting an increase in receptor translation rather than an augmentation of ER synthesis. PR level declined in the majority of the population leading to a selection of a subset of proliferating PR negative cells after treatment. These data demonstrate that the synthesis of steroid receptors is linked with the progression of cells through the cell cycle and indicate that tamoxifen blocks MCF-7 cells in G1 via its interactions with ER. Our multifluorescence imaging procedure appears to provide a rapid and quantitative approach which is useful for investigating alterations in steroid receptors after endocrine treatment.