The transcription factor RUNX2 (Cbfa1/AML3/Pebp2alphaA) is a critical regulator of osteoblast differentiation during bone formation. We investigated the effect of tumor necrosis factor alpha (TNF) on the expression of RUNX2 since TNF is known to inhibit differentiation of osteoblasts from progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 mRNA as measured by RT-PCR using primers flanking the conserved RUNT homology domain. The IC50 was 0.6 ng/ml. The effect of TNF was studied using isoform-specific primers that flanked unique regions of 2 major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting the longer MASNS isoform by 50%. TNF inhibition of RUNX2 mRNA was not blocked by cycloheximide. RUNX2 nuclear protein was also reduced by TNF, as measured by western analysis and EMSA using an osteocalcin promoter OSE2 probe. The half-life of RUNX2 mRNA was measured by RT-PCR after actinomycin D treatment. RUNX2 mRNA half-life was 1.8 HR and reduced to 0.9 HR by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6 kB RUNX2 promoter-luciferase reporter in a transient transfected assay. TNF caused a dose-dependent inhibition of RUNX2 promoter activity to 50% of control values. Deletion analysis suggested that the inhibitory effect of TNF was preserved with deletions to –108 nt upstream of the translational start site; however, localization downstream of nt –108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. Suppression of RUNX2 by TNF may decrease osteoblast differentiation and inhibit bone formation in TNF excess states.