Gelatinase B is a matrix metalloproteinase (MMP-9) produced by osteoclasts involved in bone resorption. Bone modeling, of which resorption is an integral part, is particularly evident in the intramembranous bones of the craniofacial region. To determine the role of osteoclasts in developing intramembranous bones we localized osteoclasts in calvariae from mice aged between embryonic day 16 and postnatal day 6, using gelatinase B and tartrate-resistant acid phosphatase activity (TRAP) as osteoclast markers. Through a combined approach of in situ hybridization and enzyme histochemistry, phenotypic differences between osteoclasts associated with calvarial bone were noted. Some cells expressed gelatinase B mRNA but were TRAP negative, whereas others demonstrated an overlap in enzyme profile exhibiting both TRAP activity and expressing gelatinase B mRNA. During more advanced development, most osteoclasts exhibited TRAP activity but did not express gelatinase B mRNA. The distribution of these cells differed, TRAP positive cells being detected in a widespread pattern at all ages, while gelatinase B transcripts were increasingly concentrated in areas of new and rapid bone growth, notably around the sutures. We propose the use of gelatinase B as an osteoclastic marker in the developing mouse. We conclude that gelatinase B may have a key role during early bone formation, the regulation of bone modeling, and perhaps in the maintenance of suture width.