DNA microarray based transcriptome analysis has become widely used in biomedical research; however, the lack of DNA sequence information available for Chinese hamster ovary (CHO) cells has hampered the application of microarrays for this cell line widely used for recombinant therapeutic protein production. We have constructed an expressed sequence tag (EST) based CHO DNA microarray and employed it for comparative transcriptome analysis of CHO cells and mouse hybridoma cells treated with sodium butyrate. Cross-species hybridization of CHO transcripts to mouse DNA microarrays was also performed to assess the utility of cross-species microarray. The average identity among probe sequences present on both the CHO and mouse microarray was 89.6%. Although cross-species hybridization yielded non-contradicting results when compared with the same-species arrays, decreased sensitivity was observed and resulted in fewer differentially expressed genes being confidently identified. The comparatively small number of genes probed using the CHO microarray and the low number of genes identified as differentially expressed in the cross-species hybridization limited physiological interpretation of the response of CHO cells to sodium butyrate treatment. Nevertheless, when all results are combined, mouse hybridoma and CHO cells can be seen to respond similarly to butyrate treatment, affecting histone modification, chaperones, lipid metabolism, and protein processing. To further develop the utility of microarray technology in cell culture process development, an expansion of current CHO cell sequencing efforts to increase the coverage of genes on available microarrays is warranted.