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 transcarbamoylase (OTC). When supplements were added to the medium at 33 degreesC, 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 55similar to60% higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at 33 degreesC until their growth rates approached those at 37 degreesC, 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.