1. The activation by Mg2+ of the ATP splitting of myofibrils depends on the concentration of Ca2+ ions present. If the concentration of Ca2+ is high enough to suppress the dissociation of actomyosin, Mg2+ activates, otherwise it inhibits. 2. 2. The course of the curve describing the activation by Mg2+ does not depend on the concentration of ATP present. 3. 3. In the presence of 10−4 M Ca2+, the addition of increasing concentrations of Mn2+, Ba2+, Sr2+ or Ca2+ causes an activation much resembling that caused by Mg2+. Activation is lowest when Ca2+ is the only bivalent cation present. 4. 4. Mg2+ and Ca2+ are both essential for contraction. In the presence of 2·10−5 M Mg2+ and 2·10−3 M ATP there is no contraction unless Ca2+ in a concentration sufficient to repress dissociation (approx. 10−6 M) is also present. In the presence of 10−5 M Ca2+ a maximal rate of contraction is reached only when a minimal level of Mg2+ concentration (approx. 10−6 M) is reached. There is, however, contraction in a Ca2+-free milieu too if the concentration of MgATP does not exceed 1·10−5 M. 5. 5. The substrate proper of myofibrillar ATPase (ATP phosphohydrolase, EC 3.6.1.3) is shown to be free ATP. The inhibition by a total Mg2+ concentration exceeding that of ATP is thus caused by the decrease in available substrate, i.e. free ATP. If a constant free ATP concentration is ensured there is no inhibition even by 10−2 M Mg2+.