Development of an efficient cell-free translation system from mammalian cells is an important goal. We examined whether supplementation of HeLa cell extracts with any translation initiation factor or translational regulator could enhance protein synthesis. eIF2 (eukaryotic translation initiation factor 2) and eIF2B augmented translation of capped, uncapped and encephalomyocarditis virus-internal ribosome entry site-promoted mRNAs. eIF4E specifically stimulated capped mRNA translation, while p97, a homologue to the C-terminal two-thirds of eIF4G, increased uncapped mRNA translation. When the HeLa cell extract was supplemented with a combination of eIF2, eIF2B, and p97, the capacity to synthesize a protein from an uncapped mRNA became comparable to that from the capped counterpart stimulated with a combination of eIF2, eIF2B, and eIF4E. A dialysis method rendered the HeLa cell extract capable of synthesizing proteins for 36h, and the yield was augmented when supplemented with initiation factors. In contrast, the productivity of a rabbit reticulocyte lysate was not enhanced by this method. Collectively, the translation factor-supplemented HeLa cell extract should become an important tool for the production of recombinant proteins.