Codon bias has been observed in many species. The usage of selective codons in a given gene is positively correlated with its expression efficiency. As an experimental approach to study codon-usage effects on heterologous gene expression in mammalian cells, we designed two human erythropoietin (EPO) genes, one in which native codons were systematically substituted with codons frequently found in highly expressed human genes and the other with codons prevalent in yeast genes. Relative performances of the re-engineered EPO genes were evaluated with various combinations of promoters and signal leader sequences. Under the comparable set of combinations, mature EPO gene with human high-frequency codons gave a considerably higher level of expression than that with yeast high-frequency codons. However, the levels of EPO expression varied, depending on the alternate combinations. Since the promoters and the signal leader sequences that we used are known to be equally efficient in gene expression, we hypothesized that the varied expression levels were due to the linear sequence between the promoter and the coding gene sequence. To test this possibility, we designed the EPO gene with hybrid codon usage in which the 5'-proximal region of the EPO gene was synthesized with yeast-biased codons and the rest with human-biased codons. This codon-usage hybrid EPO gene substantially enhanced the level of EPO transcripts and proteins up to 2.9-fold and 13.8-fold, respectively, when compared to the level reached by the original counterpart. Our results suggest that the linear sequence between the promoter and the 5'-proximal region of a gene plays an important role in achieving high-level expression in mammalian cells.