C6 glioma cell suspension has been inoculated into the brain of adult Long Evans rats. Animals were allowed to survive 2 to 60 days and then immunohistochemical detection of S100 protein and glial fibrillary acidic protein (GFAP) in tumors was carried out on paraffin-embedded sections. In our in vivo model the maximum positivity for both S 100 protein and GFAP was observed in C6 glial cells at 10 days after implantation. At that time increased levels of S 100 protein were expressed both in central areas containing more differentiated C6 glioma cells and in host reactive astrocytes at tumor boundary. Almost no S 100 protein was found in dividing and invading, i.e. less differentiated, C6 glioma cells at tumor periphery and in perivascular spaces of adjacent blood vessels. The distribution of GFAP positive cells followed a similar pattern as that of S 100 protein containing C6 cells. GFAP expressing cells were revealed in quiescent central tumor portions which were occupied by more differentiated cells. Tumors were surrounded by strongly GFAP positive host reactive astrocytes. Later on, when signs of tumor regression appeared there was a decrease in S 100 protein and GFAP immunoreactivity of C6 glioma cells. To summarize, we developed an in vivo model for observation of cell differentiation within a growing glioma. Less differentiated and more malignant glioma cells expressed almost no S 100 protein and GFAP in contradistinction to central and more quiescent tumor portions.