Phalloidin was shown to increase the ATPase activity and Ca z+ sensitivity of both bovine cardiac and rabbit psoas myofibrils when assayed in a solution containing 50 mM KCI, 100 mlvl MOPS (pH 7.0), 2 raM MgClz, I mM ATP, 2 mM EGTA, and varying concentrations of Ca 2รท (temperature 21-22 ยฐ C). The phalloidin effect in cardiac myofibrils developed over a time course of several minutes in the presence of 50 lxM phalloidin. Relative increase of ATPase activity was maximal at pCa 8 and decreased with decrease in pCa. In cardiac myofibrils the increase was about 70% at pCa 8 and 20% at pCa 4 following 20--30 rain pre-incubation with 2 llM or 50 I.tM phalloidin. The effect persisted after excess phalloidin was washed out. The increase in Ca z+ sensitivity was approximately 0.15 pCa units. For skeletal myofibrils treated with 2 ~tM phalloidin all changes were considerably less than those seen with cardiac myofibrils and the changes were even less when the rnyofibrils were exposed to 50 laM phalloidin. These results show that when specifically bound to actin, phalloidin can change the kinetic parameters of the cross-bridge cycle and may also alter the Ca 2+ sensitivity of the contractile system. The effects of phalloidin seem to vary with muscle type.