Messenger RNA (mRNA) expression of calpain-1 (µ-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P < 0.001) with a tendency of higher 28 kDa expression in myosin heavy chain (MyHC)-2x-rich muscles compared to MyHC-slow-rich muscles. The CSTN-I and -III expression in Longissimus thoracis (LT) showed the lowest value among the muscles tested. Moreover, 28 kDa/CSTN-I ratio was higher in the diaphragm (DP), psoas major (PM), and LT than those in the lingual muscles (TN), masseter (MS) and pectoralis (PP) (P < 0.05). Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P < 0.05). Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P < 0.05). These results indicated that the calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.