SUMMARY1. The relative isometric tension-pCa relationship has been determined for isolated bundles of barnacle myofibrils under a variety of ionic conditions using [Ca2+]-buffered solutions which also contained an ATP regenerating system (creatine phosphate and creatine kinase).2. The results are in better agreement with the 'consecutive' scheme of reaction rather than with the 'independent' alternative (Ashley & Moisescu, 1972) for the co-operative action of two Ca2+ ions in the process of tension activation in crustacean skeletal muscle.3. Variations in the pH of the activating solutions did have a marked effect on the relative tension-pCa curve, although no effect was observed on the absolute maximum value for isometric tension. A shift in pH by 0-5 u. in the range 6-6-7-6 shifted the Ca2+-activation curve by 0.5 log u. towards lower free Ca2+ concentrations.4. Changes in the free Mg2+ concentration of the activating solutions in the millimolar range produced a pronounced shift of the relative tension-pCa curve along the pCa axis. Increasing [Mg2+] from 1 to 5 mM shifted the curve by about 0-7 log u. to higher free Ca2+ concentrations, without significantly modifying its steepness.5. Changes in the MgATP concentration of the activating solutions in the range of 1-13 mm had no significant effect on the relative tension-pCa relationship.6. Varying the K+ concentration in the activating solutions was also

1. A method for producing rapid [Ca2+] and [Sr2+] changes in the frog skinned muscle fibre preparation while maintaining constant all other cationic concentrations (Moisescu, 1976a, b) is described and analysed in detail. 2. Different experiments, some of them involving the Ca2+‐sensitive photoprotein aequorin, as well as theoretical considerations, indicate that with this method one can produce a Ca2+ (or Sr2+) concentration change within 0.1‐‐0.15 sec in a whole preparation having a diameter of 50 micrometer. 3. The rate of force development was similar to that observed in vivo. 4. The radial diffusion coefficient of EGTA in relaxed myofibrillar preparations was measured and found to be 4.6 x 10(‐6) cm2sec‐1 at 20 degrees C. 5. The sarcoplasmic reticulum in myofibrillar bundles was found to be active with respect to both Ca2+ and Sr2+ in the solutions used ([Mg2+] 1 mM; [Na] 30 mM; [K] 140‐170 mM; [Cl] less than or equal to 20 mM; pH 7.10). 6. The amount of Ca released by caffeine from internal stores (previously loaded with Ca) can raise the total Ca concentration in the muscle fibre preparation by at least 1.8 mM. 7. The presence of 10 mM‐caffeine in all bathing solutions reduced drastically the ability of the sarcoplasmic reticulum to accumulate both Ca and Sr.

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5. It can be shown that, under the present experimental conditions, the Na concentration around the cells must be lower than about 0-1 mm in 'zero' Na+ solutions.6. It is shown that the over-all process leading to tension production can be explained in terms of a reaction involving two Ca ions.7. To account for the results, a modification of the reaction scheme put forward by LUttgau & Niedergerke (1958) is proposed, involving a competitive action between Ca and Na ions at a free receptor site.8. To account for the scheme, an example of a mechanism involving both internal and external Na and Ca concentrations is considered.

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