INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a “must-have” technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. The HEK293 cell line stably expressing the Epstein-Barr virus nuclear antigen-1 (HEK293-EBNA1, or 293E) is the most commonly used cell line for large-scale transfection. When using expression vectors bearing the Epstein-Barr virus origin of replication, oriP (such as the pTT vector), a threefold improvement in r-protein yield is generally obtained over a similar non-oriP vector. This protocol describes a method for culturing HEK293-EBNA1 cells which will then be used to produce recombinant proteins.