Fifty-nine paraffin-embedded astrocytic gliomas (four WHO grade 1, 21 WHO grade 2, 17 WHO grade 3 and 17 glioblastomas, WHO grade 4) were immunohistochemically investigated for expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGF-R) and oncoprotein c-erbB-2 by semiquantitative assessment. Proliferative activity was simultaneously analyzed by using the antibody Ki-67 (MIB-1). Immunostaining in neoplastic cells was quantified by image analysis. Concerning the antibodies used, the percentage of immunoreactive cells increased with histologic malignancy. There was no expression of EGF-R and c-erbB-2 in the majority of low-grade astrocytomas. However, small focal expressions of TGF-alpha and EGF-R were observed in several low-grade astrocytomas (11/25), suggesting an early stimulation of malignant transformation. With regard to percentage, a strong positive correlation between TGF-alpha and EGF-R-stained cells was found, indicating an autocrine stimulation of the mitogenic pathway of the TGF-alpha/EGF-R system. Likewise, indices of EGF-R and c-erbB-2 positive cells correlated significantly. Less significant correlations were also seen between EGF-R, c-erbB-2 frequencies and the Ki-67 labeling index. However, there was no correlation between TGF-alpha and Ki-67 indices. The results suggest that TGF-alpha expression is not directly related to the proliferative potential as judged by the Ki-67 labeling index. Furthermore, besides EGF-R and c-erbB-2, other growth factors and their receptors or mutant EGF-R might participate in the proliferative activity of gliomas.