A sandwich enzyme immunoassay method for measurement of beta subunit of muscle enolase in human serum was developed by use of purified antibodies to enolase beta subunit and beta-D-galactosidase from Escherichia coli as label. The assay was specific to the beta subunit with no cross-reaction with the alpha and gamma subunits of human enolase. The measurable range was from 10 pg to 10 ng per assay tube or 1 to 1000 ng/ml serum. Coefficients of variation within-run and between-run for the assay of serum beta subunit were less than 14%. Normal adult sera contained about 6 ng/ml of the beta subunit, and the levels were significantly elevated in sera of patients with muscular dystrophy and those with myocardial infarction. Serum levels of the beta subunit correlated well with those of creatine phosphokinase, but poorly with those of myoglobin in the same samples. The specific distribution of beta subunit in skeletal muscle and heart was confirmed by measuring the levels in various tissue extracts.