Calpastatin (CAST) is a specific protein inhibitor of the calcium dependent proteinases μ- and m- calpains. This research was performed to study the expression of CAST gene and protein in muscles and organs of Thai native cattle. Moreover, the relationship between shear force, gene expression and CAST activity in longissimus muscle of the native cattle was also studied, and compared with a native crossbred, Kampaengsaen. The CAST gene analysis was performed by RT-PCR. The results showed that the CAST gene isolated from both Thai native and Kampaengsaen crossbred was 98- 99% similar to that of the Charolais breed. The isolated fragment covered domain III and IV of the full-length gene. Relative CAST gene expression was determined in muscles and organs of the native cattle by realtime PCR using the beta-actin gene as an internal control. The results found that the CAST expression in muscles: Longissimus dorsi (LD), Longissimus troracis (LT), Transversus abdominis (TA) and Semitendinosus (ST) were approximately similar. The gene expression in Rectus abdominis (RA) muscle was twice as high as the LD whereas that in Semimembranosus (SM) muscle was 1/3 of the LD. Among the organs, the CAST gene expression in intestine and heart was 0.17-times that of LD muscle. The expression values in testis, kidney and spleen were smaller and similar between each other, while expression in brain and liver was very limited. Expression of CAST protein in muscles of native cattle was investigated by Western blot analysis using an Anti-DomIV. The protein bands corresponding to 145 and 70 kDa were found in all samples. Moreover, trace amounts of protein bands corresponding to 170, 110, 90, 60, 45 and 40 kDa were also detected. The CAST expression in RA and ST muscles was high. The expression of TA, LT and LD muscles were in small and similar amounts while that of SM was low. In tissue organs of native cattle, the CAST protein was found in heart, kidney, spleen, brain and liver, however, not detected in testis and intestine. The isoforms of 125- 145 kDa were expressed in the tissue samples. When comparing the analysis results in the LD muscles of the native and the Kampaengsaen cows, the Warner-Bratzler shear force (WBS) and the relative CAST gene expression of the native cow were significantly higher than in the Kampaengsaen cow (p<0.05). However, the CAST activities in both cows were not significantly different. The WBS, the relative CAST gene expression and the CAST activity of the LD muscles in both cows were positively correlated with p-value <0.05. This research suggested that the bovine CAST gene was highly conserved. The expressions of gene and protein depended on the type of muscles and organs. Furthermore, both gene expression and CAST activity in inhibiting the calpain enzyme had a clear effect on tenderness of the LD muscle in the Thai native cow, and to some extent also in the Kampaengsaen cow. Incorporation of gene expression and protein activity to the WBS analysis will increase the precision of the beef tenderness evaluation. The results of this research serve as fundamental information for further development of the Thai native beef quality. Moreover, it could be also applied in the medical field. This will increase both economical value and appreciation of the Thai native cattle in long term.