A sensitive sandwich enzyme immunoassay for identification of brain S-100 protein in blood or bloodstains containing brain tissue is described. A polystyrene ball coated with rabbit anti-S-100 protein IgG was incubated with human S-100 protein, and then with anti-S-100 Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human S-100 protein was 0.6 pg (30 amol) per assay tube. The cross-reaction of this sandwich enzyme immunoassay to other organs was approximately 1/100 or less. Antigenic activity of S-100 protein in bloodstains containing brain extracts was detectable after storage for 36 days at room temperature. The ratio of S-100 protein to total protein (ng/mg) in bloodstains when brain tissue was mixed with normal human blood at concentrations of 5-500 mg/ml was approximately 100-fold those of other samples (liver, heart, intestine, and skeletal muscle). These results indicated that bloodstains mixed with brain tissue were clearly distinguishable from others. Thus, in forensic practice, measurement of S-100 protein or the ratio of S-100 protein to total protein is useful to identify blood and bloodstains containing brain tissue.