The rates of osteoblast and osteoclast formation are tightly balanced, possibly due to the requirement of mesenchymal osteoblast progenitors for osteoclastogenesis. Osteoblast differentiation requires the transcription factor Cbfa1, whereas osteoclastogenesis results from the interaction between receptor activator of NF kappa B ligand (RANKL), expressed on stromal/osteoblastic cells, and RANK, a surface receptor on hematopoietic precursors. A striking decrease in the number of osteoclasts in Cbfa1-deficient mice suggested that Cbfa1 might be involved in RANKL expression. To investigate this possibility and to elucidate the mechanisms regulating RANKL expression, we isolated the 5'-flanking region of the murine RANKL gene and found that it contains two potential binding sites for Cbfa1 (OSE2-like sites). Cbfa1 bound to either of these sites in gel shift assays and stimulated the activity of a chimeric promoter consisting of multimerized RANKL OSE2-like sites inserted upstream from a minimal thymidine kinase (tk) promoter in transient transfections. However, Cbfa1 cotransfection did not stimulate murine RANKL promoter-luciferase constructs. Further analysis revealed that removal of these sites from the RANKL promoter by either site-directed mutagenesis or 5'-deletion did not alter the basal activity of promoter-reporter constructs. Conditional expression of Cbfa1 in a stromal/osteoblastic cell line stimulated osteocalcin mRNA by fivefold, but had no significant effect on RANKL mRNA levels. Conversely, conditional expression of a dominant-negative form of Cbfa1 in the same cell line inhibited osteocalcin mRNA by threefold, but had no effect on RANKL mRNA. Although these results cannot rule out a novel function for Cbfa1 in RANKL expression, they demonstrate that Cbfa1 does not regulate RANKL gene activity in the same manner as known targets of this transcription factor, such as osteocalcin.