Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase-deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2-DE with various immobilized pH gradients (pHs 3-10, 5-8, and 3-6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver-stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI-TOF-MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.