DEAE-Sephacel and phenyl-Sepharose chromatography were compared as methods for separating and quantitatively isolating calpain I, calpain II, and calpastatin from lamb muscle extracts. DEAE-Sephacel chromatography gave greater than 90% recovery of all three proteins, while phenyl-Sepharose gave only 70, 66, and 48% of the DEAE recovery of calpain I, calpain II, and calpastatin, respectively. Additionally, DEAE-Sephacel chromatography was shown to effectively separate calpastatin and calpain I. Consequently DEAE-Sephacel appears to be superior to phenyl-Sepharose for quantitative isolation of the components of the calcium-dependent proteinase system from muscle extracts. Dietary administration of β-agonist (L-644,969; Merck Sharpe & Dohme Research Laboratories) decreases extractable calpain I activity in lamb longissimus dorsi (LD) muscle by 10-14% (P < 0.05), increases calpain II activity by 34-42% (P < 0.001), and increases calpastatin activity by 59-75% (P < 0.001). Additionally, net cathepsin B activity is reduced by 30% (P < 0.05) in the LD of β-agonist-treated lambs. Reduced activity of the calcium-dependent or catheptic proteinase systems may contribute to the increased protein accretion in muscles of β-agonist-treated lambs.