Receptor activator of NF-kappaB ligand (RANKL) and interferon gamma (IFN-gamma) are critical and opposing mediators of osteoclastogenesis, exerting stimulatory and inhibitory effects, respectively. Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption. We have examined the role of IFN-gamma in the regulation of CTSK expression in the murine monocytic RAW 264.7 cell line, which can be readily differentiated to bone-resorbing osteoclasts upon RANKL treatment. Real-time RT-PCR reveals that RANKL stimulates CTSK mRNA expression in a dose- and time-dependent fashion, but that RANKL does not alter the expression of cathepsin L (CTSL) and cathepsin S (CTSS) mRNA. IFN-gamma stimulates both CTSL and CTSS expression after 3 days, but fails to significantly alter CTSK expression. IFN-gamma markedly inhibits the stimulation of CTSK mRNA and protein by RANKL, whereas RANKL suppresses the stimulation of CTSL and CTSS mRNA by IFN-gamma. IFN-gamma also ablates the RANKL induced osteoclastic differentiation of RAW cells. In RAW cells stably transfected with a CTSK promoter-luciferase plasmid containing the 1618 bp upstream of the transcription initiation site, IFN-gamma inhibits CTSK promoter activity and ablates its induction by RANKL. In conclusion, IFN-gamma and RANKL differentially regulate cathepsin K, S, and L gene expression in pre-osteoclastic cells, and there appears to be significant cross talk between the signal transduction pathways mediating the responses to RANKL and IFN-gamma. Published by Elsevier Inc.