The activation sequence of clasts (the designation clast was used because ultrastructurally in this tissue, it is not always possible to differentiate between chondroclasts sitting on cartilage and osteoclasts sitting on bone matrix) was studied in vivo using the healing of low-phosphate, vitamin D-deficiency rickets as a model system. Thus, the bones of 7-week-old rachitic animals were analyzed with a combination of morphological, biochemical, and molecular biological methods at 48 and 72 h, respectively, after change to normal food. A quantitative ultrastructural analysis showed that the number of clast profiles exhibiting the characteristic polarized features of actively resorbing cells, i.e., ruffled borders and clear zones, had reached normal levels after 48 h. By combining the data with quantitative analyses by the immunogold technique, we demonstrated that cathepsin K secretion was coupled to ruffled border formation in clasts irrespective of whether the number of polarized clasts was low (in rickets) or high (in healing). In contrast, the levels of tartrate-resistant acid phosphatase (TRAP) both between ruffles and in the outside matrix adjoining the ruffled border were low in polarized clasts both in rickets and at the early (48 h) healing time-point, but were increased at the latest (72 h) healing time-point. Interestingly, expression of TRAP and the cathepsin K at the mRNA level, as well as protein expression and the activity of TRAP, were not different during the healing sequence. Although the two enzymes are confined to the same clast populations, their secretion during the resorption process is apparently differentially regulated: cathepsin K secretion is coupled to ruffled border formation in clasts, whereas TRAP is secreted at a later stage during the resorption sequence, suggesting a role for secreted TRAP as a modulator of resorptive activity.