The Differentiation-Specific Element Binding Protein (DSEB) was identified by binding to a specific cis-acting DNA element (DSE) responsible for the irreversible continued expression of the angiotensinogen gene after differentiation of 3T3-L1 adipoblasts to adipocytes. It was also identified as the large subunit of the Replication Factor C complex. During 3T3-L1 adipoblast differentiation, DSEB is induced early and interacts with the DSE that is essential for the sustained transcriptional activation of the angiotensinogen gene. Here we describe loss of function studies in 3T3-L1 cells performed with antisense phosphorothioate oligonucleotides that hybridize to DSEB mRNA. Treatment with 15, 25, and 50 microM antisense DSEB resulted in a dose-dependent inhibition of differentiation-specific lipid accumulation after 3 days of hormonal stimulation. Similar treatment also markedly reduced differentiation-dependent expression of mRNAs encoding angiotensinogen and the fat-specific fatty acid binding protein, aP2. Further, 50 microM antisense DSEB treatment resulted in a significant approximately 50% inhibition of the cell proliferation that occurs early in 3T3-L1 adipogenesis. Control experiments using the DSEB sense oligonucleotide had no effect on hormonal-stimulated adipocyte differentiation. Combined, these results suggest that DSEB serves an important role during the proliferative phase of 3T3-L1 adipoblast differentiation.