The antitumor activity of histone deacetylase (HDAC) inhibitors has been linked to gene expression induced by acetylation of histone and nonhistone proteins; but the molecular basis for their antitumor selectivity remains largely unknown. With development of a genomically integrated, ErbB2 promoter-reporting breast cancer cell screen, ErbB2 promoter inhibiting activity was observed by the HDAC inhibitors trichostatin A (TSA) and sodium butyrate. Paradoxically, these agents stimulated the episomal form of this ErbB2 promoter-reporter introduced by transient transfection. Transcriptional run-off assays in ErbB2 amplified and overexpressing breast cancer cells confirmed that within 5 h, TSA exposure profoundly inhibits ErbB2 transcript synthesis from the amplified oncogene yet preserves transcription from single copy genes such as the epithelial-specific Ets family member, ESX. Northern analyses of ErbB2-overexpressing breast cancer lines (SKBR3, BT-474, and MDA-453) showed that within 24 h of submicromolar treatment by TSA, ESX transcript levels increase while ErbB2 transcript levels rapidly decline, with no TSA effect apparent on the open chromatin configuration of either gene as monitored by DNase I hypersensitivity. Actinomycin D studies confirmed that in addition to inhibiting ErbB2 transcript synthesis, TSA selectively destabilizes mature ErbB2 transcripts enhancing their decay. Whereas TSA markedly reduced ErbB2 protein levels in these overexpressing cell lines, TSA treatment of MCF/HER2-18 cells engineered to overexpress the ErbB2 receptor under control of a heterologous promoter increased their expression of ErbB2 protein. These findings suggest that further studies are warranted to determine whether ErbB2-positive human cancers represent unusually sensitive clinical targets for HDAC inhibitor therapy.