The pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences with the Newbler Assembler were used to achieve a major step forward to unravel the transcriptome of Chinese hamster ovary (CHO) cells. Normalized cDNA libraries originating from several cell lines and diverse culture conditions were sequenced and the resulting 1.84 million reads were assembled into 32,801 contiguous sequences, 29,184 isotigs, and 24,576 isogroups. A taxonomic classification of the isotigs showed that more than 70% of the assembled data is most similar to the transcriptome of Mus musculus, with most of the remaining isotigs being homologous to DNA sequences from Rattus norvegicus. Mapping of the CHO cell line contigs to the mouse transcriptome demonstrated that 9124 mouse transcripts, representing 6701 genes, are covered by more than 95% of their sequence length. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway of CHO cells were detected. The present manuscript represents a data set of assembled and annotated genes for CHO cells that can now be used for a detailed analysis of the molecular functioning of CHO cell lines.