Methods of fluorescence spectroscopy and microscopy-including intensity and lifetime (FLIM) images-are used to examine uptake, intracellular location and interaction of the chemotherapeutic drug doxorubicin in MCF-7 human breast cancer cells as a function of cholesterol content. By comparing cells with natural and decreased cholesterol levels after 2 h or 24 h incubation with doxorubicin, we observed that higher fluorescence intensities and possibly shortened fluorescence lifetimes-reflecting increased uptake of the drug and more pronounced drug response-are concomitant with higher membrane fluidity.