To determine the effect of low culture temperature on erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells, rCHO cells producing EPO (LGE10-9-27) were cultivated at 30, 33, and 37°C. At a culture temperature lower than 37°C cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37°C to 33°C, more than a 2.5-fold increase in the maximum EPO concentration was achieved. This enhanced EPO production at 33°C was not just because of the extended culture longevity with the decreased release of proteolytic enzymes from dead cells, but mainly because of enhanced qEPO. The qEPO at 33°C was 0.35 ± 0.08 μg/106 cells/h, which was approximately 4-fold higher than that at 37°C. Although the highest qEPO of 0.49 ± 0.14 μg/106 cells/h was obtained at 30°C, the maximum EPO concentration was lowest because the detrimental effect of lowering culture temperature on cell growth outweighed its beneficial effect on qEPO. Like qEPO, the relative EPO mRNA content increased by lowering culture temperature, indicating that the increased transcription level of EPO was responsible in part for the enhanced qEPO at low culture temperature. The quality of EPO produced at 33°C in regard to isoform pattern, sialic acid content, and in vivo biological activity was comparable to or even better than that produced at 37°C. Taken together, the results obtained demonstrate the potential of the application of low culture temperature to the commercial EPO production in rCHO cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 289–298, 2003.