The calpains, a family of calcium-requiring intracellular proteases, are proposed regulators of cell proliferation. However, ablation of the calpain small subunit gene necessary for function of the conventional calpains did not result in decreased rate of proliferative growth of mouse stem cells under routine culture conditions. To address the reasons for this discrepancy, Chinese hamster ovary cell lines were established that overexpress the calpain inhibitor protein, calpastatin, under control of the ecdysone congener, ponasterone A. Overexpression of calpastatin in these cell lines resulted in a decreased growth of isolated colonies adhering to tissue culture plates. However, when cells were plated at higher density, calpastatin overexpression had no influence on proliferative growth rate. Growth of colonies in soft agar was not inhibited by calpastatin overexpression. Cell adhesion, cell de-adhesion, and cell motility all appeared to be normal after calpastatin overexpression. Differential display analysis was initiated to detect possible alteration of gene expression upon calpastatin overexpression. Analysis of approximately 3000 differential display PCR signals resulted in identification of one band that was underexpressed. Northern blot analysis confirmed a decreased amount of approximately 1 kb mRNA in cells overexpressing calpastatin. Sequence analysis identified a putative protein, Csr, containing a region homologous to two ubiquitin transferases and a putative cation channel protein.