Tumor tissue from seven human gliomas was maintained in long-term agar overlay culture as multicellular organotypic spheroids. Light microscopic and ultrastructural observation of the spheroids displayed morphological features similar to those of the original tumor tissue in vivo; in this respect they were different from spheroids obtained from permanent cell lines. The spheroids contained preserved vessels, connective tissue, and macrophages, revealing a close resemblance to the conditions in the original tumor. Flow cytometric deoxyribonucleic acid measurements of cells from the tumor spheroids and from biopsy material obtained directly from the operation revealed the same ploidy and the same amount of proliferating cells in the spheroids as in the original tumor. Fluorescence microscopy using bromodeoxyuridine (BUdR) incorporation and anti-BUdR monoclonal antibody confirmed the proliferative potential of tumor cells in the spheroids. Diameter measurements showed that the size of the spheroids from two of the tumors increased over time while in three other cases it decreased. Spheroids from the remaining two tumors showed no change in size, even after 80 days in culture. These growth data and the relatively high number of proliferating cells, as measured by flow cytometry, indicate that the degree of cell proliferation and cell loss from the spheroids are closely linked, as is the case for tumors in vivo. The culture system presented provides a valuable alternative to propagation of human tumors in animals.