Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHODG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of -2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHODG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells. Biotechnol. Bioeng. 2008;101: 182-189. (C) 2008 Wiley Periodicals, Inc.