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Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions. The average insert size for both libraries is approximately 800-850 bp, and each library has comparable redundancy levels of 36%-38% for the sequences isolated. Random sequencing of 4,608 ESTs yielded 2,602 unique assemblies, 76% of which were annotated as orthologs of sequences in the GenBank database. A high abundance of mitochondrial genome transcripts facilitated the assembly of the complete mitochondrial genome by PCR walking. Comparative analysis of sequences from both mitochondrial and nuclear genomes with orthologous genes from other species shows that CHO sequences are generally most similar to mouse; however, examples with highest similarity to rat or human are common. A cDNA microarray, including all 4,608 ESTs, was constructed. The microarray results reveal a high level of consistency between transcript abundance in the libraries and fluorescence intensities. Inclusion of redundant clones in the microarray, additionally, allows small changes in abundant mRNAs to be discerned with a high degree of confidence. The information and tools generated provide access to genomic technology for this important cell line.