By hybridizing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with the glial cell-rich fraction prepared from an autopsied human brain, we established a hybridoma that produces a monoclonal antibody to protoplasmic astrocytes (PA). The antibody, named PRAS-1, consistently labeled cytoplasm of PA with a granular pattern. In a few cases, the cytoplasmic processes of several astrocytes in gray and white matter were also stained. The immunoreactivity was lost after periodic acid treatment or methylation, showing that the epitope is composed of a carbohydrate. The cytoplasmic reaction was resistant to protease digestion and lost after incubation in an organic solvent, suggesting that a glycolipid is the antigen. On the other hand, the reaction in the processes disappeared upon protease digestion. Ultrastructurally, the immunoreaction was localized to secondary lysosomes. Cross-reactivity was noted on a small number of incidental neurons, corpora amylacea, hepatocytes and esophageal epithelial cells. A long period of formalin fixation did not deteriorate the antigenicity. PRAS-1 was demonstrated to detect PA immunohistochemically on paraffin sections, and may be applicable to further investigations into development or neoplasms of human astrocytes.