Immunocytochemistry using antibodies against various molecular forms of the Ca++ and Zn(++)-binding S100 proteins predominantly labelled astrocytes. However, especially in the neocortex the staining pattern is variable. Methods of tissue preparation have been evaluated with the aim to preserve as much S100 immunoreactivity as possible. Optimal results were obtained after perfusion fixation with 4-5% aldehydes, 0.1 M sodium cacodylate, 0.1% CaCl2, pH 7.3. In such preparations, astrocytes were completely labelled including their lamellar compartments in large parts of the central nervous system. Ca(++)-withdrawal had adverse affects on S100 immunoreactivity. Cryostat sections treated with EDTA-containing solutions before fixation showed that Ca(++)-free S100 can apparently not be fixed to the tissue. Perfusion fixatives containing EDTA resulted in inhomogeneous loss of S100 staining, indicating a differential susceptibility of astrocytic subpopulations. A different type of reduction in S100 immunoreactivity occurred around large neocortical blood vessels. Perivascular defects in immunostaining occasionally appeared even after optimal fixation, but could be regularly provoked by mildly acidic fixation (pH 6.6) or prolonged barbiturate anaesthesia. These defects might be based on S100 release into the cerebrospinal fluid. Presumably under none of the conditions studied can the immunoreactivity of all S100-forms and -fractions be completely preserved in the tissue. However, recommendations are presented for optimizing tissue preparation, to the extent that premortal modifications affecting the stainability of astrocytes may be detected by S100 immunohistochemistry in fixed brain tissue.