Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.