This semi-automated colorimetic assay for the MB isoenzyme of creatine kinase (EC 126.96.36.199) is based on a monoclonal antibody ('Conan-MB') specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986; 32: 657-63). A 0.64-cm bead coated with 2 to 3 μg of antibody is incubated with 100 μL of serum and 10 μL of 0.2 mol/L β-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37°C. A solution containing trans-1,2-diaminocyclohexane-N, N, N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB ≥ 12 U/L (n = 347) were verified by electrophoresis on agarose.