An in vitro test of cell sensitivity to drugs that indicates in vivo response is an important need in cancer therapy and cancer drug development. Toward this end, we previously developed a collagen gel-supported culture system for growth of human tumors. This three-dimensional culture system is general and grows tumors at high frequency directly from surgery or biopsy that maintain important in vivo properties in vitro, including tissue architecture. We report here that with autoradiographic techniques measuring cellular DNA synthesis the drug responses of individual cells within the tissue structure of in vitro-grown tumors can be determined. Twenty tumor classes, including all the major ones, have been measured in toto at >50% frequency. Quantitative and qualitative results show increasing cell kill with rising cytotoxic drug concentration, differential drug sensitivities of multiple cell types within individual cultured tumors, differential sensitivities of a series of tumors of the same histopathological classification to a single drug, differential sensitivities of individual tumors to a series of drugs, and sensitivity patterns of various tumor types similar to the sensitivities found in vivo.Therefore, the results indicate that potentially important therapeutic data can be obtained from tumor specimens growing in vitro for the individual cancer patient as well as for rational and relevant screening for new agents active against human solid tumors.A major clinical problem is that cancers that are classified as identical according to their histopathological characteristics are nonetheless highly individual in their drug sensitivities and there is currently no way to predict clinical outcome of chemotherapy for individual patients (1). A second major problem is that there is currently no relevant assay to screen for new human anticancer agents, especially for solid tumors (2). To overcome these problems, many attempts have been made to develop in vitro drug-sensitivity tests for individual cancer patients about to undergo chemotherapy and to screen for new anticancer agents. These attempts have suffered in one form or another from their inability to support growth of human tumors such that they reflect the in vivo situation. Plating of dissociated tumor cells in soft agar (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and monolayer cultures (13-15) does not in many instances allow the growth of tumor cells. Often, when tumor cells do grow under these conditions, other cell types present in the original tumor probably have not grown. It is critical that cell types present in the original tumor be present in the assay since it has been shown that interactions between cell types can alter their drug sensitivities (16). Multicellular spheroids that are three-dimensional have been used recently for drug sensitivity testing (17,18), but these also involve dissociation of cells from the tumor and reassociation into structures that do not resemble the original tissues. Short-term in vitro assays of drugs on noncultured n...