The chronology of appearance of calpain I and calpain II was analyzed during myogenesis of embryonic myoblasts in culture. The influence of the hormones insulin and corticosterone, and insulin growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-beta) on the modulation of calpain-calpastatin levels during myogenesis was also analyzed. Immunodetection assays using specific antibodies and enzymic activities showed that during muscle cell differentiation in vitro, calpain II is present from the beginning of myoblast fusion (2nd day) increasing until the 6th day and then reaching a plateau. These observations were confirmed by an analysis of the expression of total calpain mRNAs which followed the same time profile, thereby providing evidence for a transcriptional regulation in the expression of calpains. Even if an increase in calpain II activity occurs at approximately the same time as an increase of fusion, calpain II activity and rate of fusion are not closely correlated. The involvement of calpain II in some event that follows myoblast fusion is suggested. On the other hand, calpain I and calpastatin were detected only on the 6th day of cell culture growth; these results enable us to argue that if calpain I has any biological role (which remains to be established), this role occurs during the final stages of muscle cell differentiation. The presence of exogenous factors which are known to affect muscle cell differentiation by altering either the rate of protein synthesis, or degradation or both, significantly affects the modulation of calpain-calpastatin levels. Such a regulation at the transcriptional level suggests that calpains do not act as housekeeping enzymes during myogenesis.