A brain tumor is composed not only of tumor cells, but also of normal glial, mesenchymal, endothelial, and microglial cells, as well as lymphocytes and macrophages. Therefore, homogeneous cultures of tumor cells, currently used for chemosensitivity testing, do not accurately model in situ tumors. We have developed an in vitro growth assay for brain tumors that includes normal host cells and is potentially useful for studies of chemotherapy and biological response modifiers. Human glioblastoma xenografts (U251-MG) were resected from mice, minced, and explanted into agarose-coated culture wells. After 5 to 7 days, microtumors emerged as expanding spheroids, which grew most efficiently in minimum essential medium supplemented with 20% fetal calf serum, 90% of which was replaced on alternate days. The growth rate and bromodeoxyuridine labeling index were similar in the microtumors and the xenografts, and light microscopy revealed highly cellular, pleomorphic tumors with high mitotic activity in both. Immunohistochemical studies also demonstrated the persistence of macrophages in both xenografts and microtumors. Microtumors treated for 2 hours with 75 mumol/L 1,3-bis-(2-chloroethyl)-1-nitrosourea showed a growth delay of 1.5 days; no effects were observed after treatment with lower doses. This in vitro system for brain tumor culture may provide a useful technique for the study of new therapies as an alternative to in vivo xenograft studies using immunodeficient animals.