The interactions of tamoxifen with lipid bilayers of model and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py(3)Py). The effects of TAM of liposomes of DMPC, DPPC and DSPC are temperature dependent. In the fluid phase, TAM reduces dynamics of the upper bilayer region as observed by Py(3)Py and has no effect on the hydrophobic region as detected by DPH. In the gel phase, the effects of TAM evaluated by Py(3)Py are not discernible for DMPC and DPPC bilayers, whereas DSPC bilayers become more fluid. However, DPH detects a strong fluidizing effect of TAM in the hydrophobic region of the above membrane systems, where DPH distributes, as compared with the small effects detected by Py(3)Py. TAM decreases the main phase transition temperature but does not extensively broaden the transition thermotropic profile of pure lipids, except for bilayers of DMPC where TAM induces a significant broadening detected with the two probes. In fluid liposomes of sarcoplasmic reticulum lipids and native membranes, TAM induces an ordering effect, as evidenced by Py(3)Py, failing DPH to detect any apparent effect as observed for the fluid phase of liposomes of pure lipid bilayers. These findings confirm the hydrophobic nature of tamoxifen and suggest that the localization and effects of TAM are modulated by the order and fluidity of the bilayer. These changes in the dynamic properties of lipids and the non-specific interactions with membrane lipids, depending on the order or fluidity of the biomembrane, may be important for the multiple cellular effects and action mechanisms of tamoxifen.