Receptor activator of NF-κB ligand (RANKL) has been identified as a prerequisite for osteoclastogenesis. To elucidate the molecular mechanism of osteolytic bone lesions, we investigated RANKL mRNA expression in mouse bone tissue from experimental models of mouse autoimmune arthritis and cancer associated osteolysis. Elbow joints of type II collagen-induced arthritis (CIA) mice and calvariae of mice transplanted with breast cancer cells under the scalp were fixed with 4% paraformaldehyde for 2 days, decalcified with 20% EDTA in phosphate buffer for 5 days and embedded in paraffin. For in situ hybridization, digoxigenin-labeled antisense (or sense) single-stranded DNA probes were prepared by unidirectional PCR using cDNA of mouse RANKL amplified by RT-PCR. In the articular lesions of CIA mice, TRAP-positive osteoclasts were found at eroded cartilage and bone as well as in the pannus. Synovial cells around these osteoclasts were strongly positive for RANKL, indicating that synovial cells contribute to osteoclastogenesis through authentic RANKL-RANK signaling. In the tumor-induced osteolytic lesions of the mouse calvaria, osteoblasts and stromal cells between the tumor foci and the bone surface expressed RANKL mRNA; osteoclastic bone resorption was observed adjacent to the RANKL-positive cells. Histological evaluation of RANKL expression in bone lesions is important, because osteoclasts and the RANKL-RANK system could be potential targets for therapeutic drugs in diseases with accelerated bone resorption.