The purpose of study was to investigate the effects of sample handling during chromatographic separation and frozen sample storage periods on activities of calpain I and II and calpastatin in bovine skeletal muscles. DEAE-Sephacel chromatography was used to seperate the calpain I and II and calpastatin. They were eluted from the column by a continuous gradient procedure (linear gradient of NaCl from 100 to 500 mM NaCl in equilibrating buffer) and a stepwise gradient procedure (stepwise gradients of 100, 200, and 400 NaCl in equilibrating buffer). Compared to the continuous gradient elution, the stepwise gradient elution provided easy and convinient way of separation of calpain I and II and calpastatin because gradient former and pump were not required for collecting fractions. `The activities of calpain I and B did not change during frozen storage period of samples at -70℃ for 0, 2, and 4 weeks, but the activity of calpastatin gradually decreased (p$lt;0.05) during the period. The activities of calpain I, calpain II and calpastatin in fractions decreased gadually (p$lt;0.05) during storage at 4℃ for 0, 7, and 14 days. Calpastatin was heat-resistant, and the activity of calpastain in boiled fraction increased by 7%. The results of this study indicate that 1) to measure the accurate activity of calpastatin in vivo, fresh samples, not frozen, should be used, 2) the activities of calpain I and II and calpastatin should be measured rapidly after the fraction collection from the column.