Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcar-bamoylase (OTC). When supplements were added to the medium at $33;^{circ}C$, the growth of IBE and CO5 cells increased by $27\%;and;26\%$, respectively and the maximum yield of EPO was increased by $40\%$ in both cell lines. The absolute EPO concentration in the CO5 cells was always $55{sim}60\%$ higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at $33;^{circ}C$ until their growth rates approached those at $37;^{circ}C$, the growth rates of both IBE and CO5 cells increased by $54\%$ and their maximum EPO levels increased by up to $73\%;and;56\%$, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.