This experiment examined the effect of feeding a concentrate diet ad libitum for two periods of time in two breeds of contrasting mature size cattle, on the activity of the components of the calpain system and the association between components of the calpain system and tenderness as determined by shear force in the longissimus muscle. Nine-month-old Angus and Simmental steers were either fed ad libitum on a feedlot from 9 months of age (F9) or on pasture until 25 months of age (F25) and then fed on the same ad libitum ration as the F9 steers. Groups within a breed were killed at mean liveweights of c. 800 kg for the Angus and 950 kg for the Simmental. Steers were slaughtered in a commercial abattoir and muscle samples collected from the longissimus muscle at the 13th rib within 60 min of slaughter. The activities of the components of the calpain system were determined 2 and 24 h post-slaughter. The length of time on the feedlot diet had the largest effect on shear force of the longissimus muscle and the activity of μ-calpain. The steers growing fastest prior to slaughter (F25) had the highest μ-calpain activities two h post-slaughter and produced the most tender meat, even though they were slightly older at slaughter. The best component of the calpain system for predicting shear force over all the treatments was the activity of calpastatin 24 h post-slaughter. This association between shear force 21 days post-slaughter and the activity of calpastatin 24 h post-slaughter was weak and varied between the treatments. The strongest associations occurred in the F9 Angus steers who also had the toughest meat and the slowest growth rates prior to slaughter.