We studied the regional distribution of proliferating tumor cells in five avian sarcoma virus-induced gliomas. The labeling index and spatial distribution of [3H]thymidine (dThd)-labeled tumor cells were determined in serial sections of each tumor with a computer-assisted digitizing system. The density of [3H]dThd-labeled cells showed marked regional variation in each tumor, and the ratio of the density of [3H]dThd-labeled cells in tumor periphery to tumor center varied from 0.86 to 1.38. The labeling index generally, but not always, reflected [3H]dThd-labeled cell density. This study indicates that proliferating pools of glioma tumor cells exhibit regional variability in concentration and that the highest numbers of proliferating cells may be predominantly located in central regions of tumor and not in tumor periphery as assumed previously. In all tumors, large numbers of proliferating cells were present in all parts of the tumor.