Tartrate-resistant acid phosphatase (TRAP) is a standard histochemical marker of differentiated osteoclasts and has been proposed as a serum/plasma marker for osteoclast activity. Enzyme assays have been described that show elevated TRAP enzyme activity in the serum or plasma of patient groups known to have increased bone metabolism. However, the poor stability of the enzyme and potential contribution from nonosteoclastic sources make it problematic to measure in patient samples. Immunoassays developed to measure TRAP in serum and plasma have yielded widely varying TRAP levels in both normal and disease states. It is not clear if this variability is caused by differences in assay calibration, antibody specificity, and/or TRAP instability. In this paper, we report that purified TRAP spiked into serum forms high molecular weight complexes. Complex formation results in greatly decreased TRAP enzyme activity and immunoreactivity. The complexing protein in serum has been identified as alpha2-macroglobulin (alpha2M). Similar complexes are observed in stored patient samples. In vitro studies with purified components show that TRAP binds to alpha2M primarily through noncovalent ionic interactions. Our results demonstrate that one mechanism of TRAP instability in serum is complex formation with alpha2M and suggest further that current TRAP enzyme and immunoassays may not accurately measure the circulating level of TRAP.