Although a number of methods for identification of human blood or bloodstains and tissue fragments that used enzyme immunoassay have been reported, immunoelectrophoresis of human beta-enolase suggests that it shows species specificity. The authors, therefore, theorized that it would be possible to discriminate blood, bloodstains, and tissue fragments of human origin from those of other species, as well as to identify skeletal muscle injury. A 33-year-old man was hit and run over by a car. The car involved was found the next day, and a minute quantity of a dried substance that appeared to be a tissue fragment was found adhering to the bottom of the car. Ten mg of this substance was homogenized with 1 ml of NaP buffer, and extract was obtained. The origin of the substance was examined by using a sandwich enzyme immunoassay. The study found that human beta-enolase is detectable in human blood at dilutions up to 3x10 cubed and human muscle extract (0.1 g/ml) at dilutions up to 5x10 (to the sixth power) using a sandwich enzyme immunoassay. The cross-reactivities of both blood and skeletal muscle extracts from other species were 0.01 or less. When human skeletal muscle extract was added in a final concentration of 10 mg/ml, the ratio of beta-enolase to total protein in bloodstains was approximately 1,000; whereas, the addition of skeletal muscle extract of other species resulted in a ratio of less than 10. Application of this method in a practical case proved that human muscle-specific beta-enolase as a marker for species identification is effective in forensic practice. 2 figures and 15 references