Ca2+-activated Z-disk-removing activity in the P0–40 crude muscle extracts described by Busch et al. (Busch, W. A., Stromer, M. H., Goll, D. E., and Suzuki, A. (1972), J. Cell Biol. 52, 367) was purified from porcine skeletal muscle extracts by using five column chromatographic procedures in succession: (1) 6% agarose; (2) DEAE-cellulose; (3) Sephadex G-200; (4) DEAE-cellulose with a very shallow gradient; (5) Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca 2+-activated proteolytic activity, so Z-disk-removing activity in the P0–40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The five column chromatographic procedures produced a 140-fold increase in specific activity of the Ca 2+-activated proteolytic enzymic activity; because preparation of the P0–40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure described here produces a 17 800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 μg of CAF per g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25–0.76 μg of purified CAF per g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HCl buffer but migrated as two bands with molecular weights of 80 000 and 30 000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80 000- and 30 000-dalton subunits make up 85 to 90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.