The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNER-Fc). After screening several promoter and enhancer combinations for high levels of TNER:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2 x 10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNER-Fc yields over 150 mu g/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300 mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells. (C) 2015 Elsevier B.V. All rights reserved.