Ah~tract-To obtain further evidence concerning the intracellular action of ouabain, we observed the effect of this compound on contractures evoked by rapid cooling in the presence of caffeine (rapid cooling caffeine contraeture; RCCC), in cardiac muscles, under conditions of a Ca deprived medium. The experiments were carried out at 20 C using cat papillary muscles and frog ventricle strips, with the exception of cooling (2`C). The muscles were first immersed in normal solution, electrically driven, then immersed in 1 mM EDTA solution for 6 min. The strips were then immersed for 10 min in a caffeine solution prepared by eluting a no-Ca solution containing 50 mM caffeine through Dowex A-I, Ca-chelating resin. Finally, the caffeine solution was exchanged with fresh caffeine solution of 2-C to evoke the RCCC. Ouabain (1 10-7 1 x 10 M) markedly potentiated RCCC. An ouabain-induced increase of RCCC did not appear in the resting strips unless the tissues were electrically stimulated.'H ouabain occupied a considerable cellular space (0.55) at the appearance of the ouabain potentiation of RCCC (''C-inulin space; 0.20). The appearance of the ouabain potentiation of RCCC was independent of changes in 22Na, Ca2+ and ATP contents in the strips. A possible mechanism of the potentiating effect of ouabain on con traction is discussed.In view of recent studies on the excitation-contraction (E-C) coupling mechanism in the heart and skeletal muscles (1-5), there are several possible mechanisms underlying the positive inotropic action of cardiac glycosides on the heart muscle. Lee and Klaus (6) and Nayler (7,8) concluded that the inotropic response to the glycosides reflects the effects on E-C coupling, and not the effect on either energy metabolism or the contractile protein.According to their results, an increase of Ca-influx following drug application to the heart muscle is an essential factor for the appearance of their positive inotropic actions. We have reported that in crab single muscle fibers contractile responses (twitch, potassium-induced contracture and caffeine-induced contraeture) are remarkably potentiated, if ouabain is microinjected into the fiber; whereas, the situation is reversed if the drug is given extra cellularly (9). Recently it was reported that ouabain potentiation was produced without a change of inward calcium currents in cat myocardium (10). The present communication deals with this observation on both cat and frog cardiac muscles in which there was evidence of a positive inotropic action of ouabain without the involvement of extracellular Ca, namely,