The mechanism of contraction in rabbit fast-twich, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponin-tropomyosin regulated actin heavy meromyosin. The Ca2+ and Sr2+ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca2+ or Sr2+. By contrast, rabbit adductor magnus (fast-twich) skinned fibers required substantially higher [Sr2+] than [Ca2+] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr2+ than Ca2+ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca2+ and Sr2+ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca2+- and Sr2+-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.