Human cancer cell lines grown in vitro are frequently used to decipher basic cell biological phenomena but also to specifically study different forms of cancer. Here we present the first large-scale study of protein expression patterns in cell lines using an antibody-based proteomics approach. We analyzed the expression pattern of 5436 proteins in 45 different cell lines using hierarchical clustering, principal component analysis and two-group comparisons for the identification of differentially expressed proteins. The results show that protein profiles of cell lines, as determined using immunohistochemistry, allow for a hierarchical clustering that overall reflects tumor tissues of origin. Hematological cell lines appear to retain their protein profiles to a higher degree than cell lines established from solid tumors, resulting in a clustering that well reflects progenitor cell types. The discrepancy may reflect different levels of in vitro induced alterations in adherent and suspension grown cell lines, respectively. In addition, multiple myeloma cells and cells of myeloid origin were found to share a protein profile, relative the protein profile of lymphoid leukemia and lymphoma cells, possibly reflecting their common dependency of bone marrow microenvironment.