Immobilization causes a transient increase in bone resorption and a prolonged depression of bone formation. We have studied the effect of immobilization on the expression of two proteins believed to have a major functional role in osteoclasts, the proteolipid subunit of vacuolar H⁺‐ATPase (VPL) and carbonic anhydrase II (CA II). Trabecular bone from immobilized rat tibiae was analyzed using northern and slot blotting, polymerase chain reaction (PCR), and morphometric analysis. CA II and VPL transcription was rapidly stimulated in trabecular bone of immobilized rat tibiae. Osteoclast number increased and the trabecular bone volume decreased during immobilization. Fluorescein‐labeled cDNA probes and a confocal laser scanning microscope were used to study the localization of VPL and CA II mRNAs in situ in osteoclasts and other bone‐derived cells attached to bovine bone slices in vitro. CA II and VPL mRNA were highly expressed in actively resorbing osteoclasts, but in nonresorbing osteoclasts mRNA expression was very low or not detectable at all. These results strongly suggest that both CA II and VPL have an important functional role in bone resorption. Finally, in the bone cell population isolated for these studies, CA II was found to be highly specific for osteoclasts whereas VPL was also detected in other cell types.