A sandwich enzyme immunoassay system for detecting tenascin in human serum was established using purified antibodies to tenascin. The assay system comprises polystyrene balls with immobilized polyclonal antibody F(ab')2 fragments and monoclonal antibody F(ab')2 fragments labeled with beta-D-galactosidase from Escherichia coli. The assay system has a minimum detectable sensitivity of 10 ng/ml of tenascin in human serum, with an assay range of 3 micrograms/ml. The assay system was found not to cross-react with laminin, vitronectin, human epidermal growth factor, fibrinogen, or fibronectin. Coefficients of variation within-run and between-run for the assay of human serum tenascin were less than 10%. Serum samples from healthy adults (n = 86) contained about 800 ng/ml and serum tenascin concentrations of patients with carcinoma (n = 47) were increased. These results suggest that tenascin in serum might be a marker substance for carcinoma.