Alkaline phosphatase (ALP) is present in human serum in the form of several isoenzymes. The two major circulating ALP isoenzymes, bone and liver, are difficult to distinguish because they are the products of a single gene and differ only by posttranslational glycosylation. Quantitative measurement of bone ALP (BAP) activity in serum can provide an index for the rate of bone formation. Furthermore, increased BAP activity in serum is indicative of bone disorders. We describe a method in which serum samples are added to a microtiter plate coated with monoclonal anti-BAP antibody and incubated 3 h at room temperature. After the unbound materials are washed off, the bound BAP activity is measured by adding p-nitrophenyl phosphate substrate. The assay demonstrated no cross-reactivity to intestinal or placental ALP and only 3-8% cross-reactivity to liver ALP. The intraassay (n = 21) CVs were 3.9-5.9%, and interassay (n = 8) CVs were 4.4-7.0%. Comparisons of the assay (y) with an IRMA (x) and a wheat germ agglutinin precipitation method (x') gave regression equations of y = 1.32x-6.4, r = 0.99, and y = 1.41x' + 4.8, r = 0.99. The assay detected increased BAP in sera from patients with osteoporosis, Paget disease, osteomalacia, or primary hyperparathyroidism.