Pax5 plays a key role in the progression of B cell development. Its expression is observed in a wide range of cell types from early lineage-committed precursors up to mature B cells, but is silenced in terminal differentiated plasma cells. In this report, we show that DNA methylation is involved in the silencing of Pax5. In the Pax5-expressing cell lines 38B9 (pre-B) and 2PK-3 (mature B), all CpG sites in TATA-containing upstream promoter were unmethylated, whereas these sites were completely methylated in myeloma cell lines FO and Sp-2/0, which do not express Pax5. Demethylation of FO and Sp-2/0 with 5-aza-2′-deoxycytidine (5-aza-dC) resulted in Pax5 re-expression with the concomitant expression of CD19 and mb-1 genes, which are known to be the target genes of Pax5. Re-expression of Pax5 was also induced by trichostatin A (TSA), which was a specific inhibitor of histone deacetylase. This re-expression was, however, transcribed only from the TATA-less downstream promoter. Taken together, we concluded that the upstream promoter was predominantly inactivated by DNA methylation, while the downstream promoter was repressed by the histone deacetylation. This synergetic inactivation of two promoters results in the final silencing of Pax5 expression in terminally differentiated B cell lines.