We have recently reported the identification of a novel member of the leukocyte receptor family, osteoclast-associated receptor (OSCAR), which has two Ig-like domains and functions as a bone-specific regulator of osteoclast differentiation. Here, we have cloned the OSCAR promoter region to examine its regulation by transcription factors. The 1.7-kb promoter region of the mouse OSCAR gene contains two potential E-box elements for microphthalmia transcription factor (MITF) and three putative PU.1 sites. MITF or PU.1 alone activates the OSCAR reporter construct 5-6-fold, and the combination of MITF and PU.1 synergistically activates the OSCAR reporter activity up to 110-fold. The mRNA expression patterns of MITF, PU.1, and OSCAR in TRANCE-treated (RAW 264.7) or TRANCE/M-CSF-treated cells (primary osteoclasts) reveal that MITF mRNA expression is induced at a much earlier time point than OSCAR gene expression. In contrast to MITF, PU.1 mRNA levels remain relatively constant at all time points, suggesting that TRANCE-induced MITF, not PU.1 expression, is one of the critical regulatory mechanisms for optimal OSCAR expression during osteoclastogenesis. In addition, we have shown that the combination of MITF and constitutively active MKK6-expressing plasmids synergistically activates OSCAR reporter activity. Taken together, our results strongly suggest that PU.1 and MITF transcription factors synergistically activate OSCAR gene expression. Moreover, the activation of OSCAR gene expression by PU.1/MITF is further enhanced by the TRANCE-induced MKK6/p38 signaling cascade.