CCAAT/enhancer-binding proteins (C/EBPs) are involved in the regulation of cell proliferation, differentiation, and control of metabolic function. Although the roles of C/EBPs in osteoblasts are largely unknown, both C/EBPβ and -δ have been shown to enhance rat osteocalcin promoter activity through the synergistic activation of Runx2 at the C/ EBP element. Here we show that in the mouse, C /EBPδ increases the expression of osteocalcin whereas C/EBPβ does not. This increased expression was found to occur at the transcriptional level, as demonstrated by the increased transcriptional activity from mouse osteocalcin II (OG2) promoter by C/EBPδ. Although we found three putative C/EBP sites in the -637/±34 region of the OG2 promoter, none of these sites showed binding activity with in vitro translated C/EBP proteins. Notably, we show that C /EBPδ physically interacts with Runx2 and that C/EBPδ overexpression increases binding between the Runx2-C/ EBPδ complex and the OSE2 element, a critical osteoblast-specific cis-acting element in the OG2 promoter. Consistent with these DNA binding data, a mutation in OSE2 abrogated the stimulatory effect of C/ EBPδ on this promoter activity. Finally, chromatin immunoprecipitation analysis in MC3T3-E1 cells showed in vivo occupancy of the OG2 promoter by Runx2 and C/EBPδ. In conclusion, C/EBPδ was found to regulate mouse osteocalcin OG2 promoter activity indirectly by interacting with Runx2 in the context of the OSE2 element and this subsequently resulted in the cooperative activation of the OG2 promoter.