Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. A biodegradable, biocompatible and non-toxic polymer chitosan was employed for 3D culture of MCF-7 cell lines. Cells grown on chitosan scaffold produce more lactate from glucose in comparison to that secreted by cells grown on tissue culture plate, thus indicating the suitability of chitosan scaffold as an in vitro model resembling cancer tissue growth in vivo. Cytotoxic effect of tamoxifen at different concentrations was evaluated for MCF-7 breast cancer cell lines grown on tissue culture plate as well as on 3D chitosan scaffold. At a tamoxifen concentration of 10(-6) M, 50% reduction in cell growth was observed in tissue culture plate-grown cells where 15% reduction in cell growth was observed when cells were grown in chitosan scaffold. Higher tamoxifen concentrations were required to achieve comparable cytostatic action in 3D culture, supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. Carbohydrate metabolism of MCF-7 cells in terms of glucose utilization and lactate production in 3D and monolayer culture were unaffected by tamoxifen treatment. Cathepsin D activity, an autocrine growth factor in breast cancer cells was monitored in all experiments. In 3D culture, addition of tamoxifen promoted cathepsin D secretion but inhibited its uptake by cells. Growth of cells in 3D chitosan scaffold indicated that action of tamoxifen on estrogen positive cancer cells is also mediated through inhibition of cathepsin D uptake from the culture medium.