Alexis Carrel introduced the in vitro culture of tissues in the beginning of the century utilizing a culture system that allowed the three-dimensional growth of tissues. Leighton improved upon this system by developing a substrate of sponge matrices. Other methods of three-dimensional culture include collagen gels and what are known as organ culture systems on filters or meshes. In addition, cell suspensions can be converted into multicellular spheroids, another form of three-dimensional culture. Comparison of the three-dimensional culture methods with two-dimensional culture methods has shown critical differences in the behavior of biological systems in culture. For example, in vivo-like drug responses are observed in three-dimensional but frequently not in two-dimensional cultures, indicating that drug response may be a function of tissue architecture. The in vivo mechanism of drug resistance may involve alterations in cell-cell interaction which may occur in three-dimensional culture as opposed to monolayer culture. Practical applications of three-dimensional culture include the development of a drug-response assay that correlates not only with drug resistance but also with drug sensitivity and survival of cancer patients. It has been shown that gene expression may be more in vivo-like in three-dimensional cultures than in two-dimensional monolayer cultures. For example, tumor antigens may be expressed in three-dimensional culture and not in monolayer culture. Thus, future studies utilizing three-dimensional cultures may significantly enhance our understanding of gene expression and resistance to drugs and enhance the efficacy of cancer chemotherapy by correctly predicting active drug regimens for individual patients.