Human serum contains two forms of tartrate-resistant acid phosphatase (TRAP), 5a and 5b. Of these, 5a contains sialic acid and 5b does not. We show here that antigenic properties and pH optimum of TRAP purified from human osteoclasts are identical to those of serum TRAP 5b and completely different from those of serum TRAP 5a, suggesting that 5b would be derived from osteoclasts and 5a from some other source. We developed a novel immunoassay specific for 5b using a monoclonal antibody O1A as capture antibody. O1A did not bind acid phosphatase derived from platelets and erythrocytes. Western analysis showed that O1A was specific for TRAP in both human bone and serum. We measured bound TRAP activity at pH 6.1, where 5b is highly active and 5a almost completely inactive. The immunoassay detected more than 90% of the initial TRAP 5b activity after 8-h incubation of serum samples at 25 degrees C and after 3 days incubation at 4 degrees C. Serum TRAP 5b activity decreased significantly after 6 months of hormone replacement therapy (HRT) of postmenopausal women compared with the change observed in postmenopausal women receiving placebo (p < 0.0001). Instead, no significant differences were observed between the changes in the placebo and HRT groups in total serum TRAP amount. These results show that serum TRAP 5b is a specific and sensitive marker for monitoring antiresorptive treatment. Instead, total serum TRAP cannot be used for that purpose. These findings may turn out to be a significant improvement in using serum TRAP as a resorption marker.