Fast, glycolytic muscle fibers are typically subjected to stimulation of short duration and high intensity. In response, force reversibly declines; i.e. the fibers fatigue. In well-motivated subjects, the cause of this type of fatigue resides within the muscle rather than in the central nervous system, peripheral nerves, or neuromuscular junction. Therefore, it has been appropriately studied in isolated muscle fibers. The cellular basis for fatigue is not yet well understood, partly because models have been constructed from studies on a wide range of species, preparations, conditions, and fatiguing protocols that consider only restricted aspects of the problem. Ca2+ uptake and release by the sarcoplasmic reticulum (SR) and the response of the contractile apparatus to the released Ca2+ are likely sites at which impairment may produce fatigue. What is the immediate cause of fatigue; what is the agent or agents that directly affect the SR and contractile proteins? This question has not as yet been answered. Our working hypothesis is that the changes in contractile force associated with fatigue and recovery from fatigue are paralleled by changes in the intracellular milieu and that these changes are causative. To test this hypothesis, we will carry out a two part study: 1) We will measure the intracellular concentration of the constituents of the milieu that we think most likely cause fatigue, under the following conditions: i) control; ii) during fatigue; iii) with recovery from fatigue. The constituents are: a) ATP, AMP, IMP, creatine, lactate, inorganic phosphate, phosphocreatine, glycogen, and H+ by biochemical analysis; b) K+ and Na+ by flame photometry; c) inositol (1,4,5)trisphosphate by specific binding assay; d) cAMP by radioimmunoassay; e) Mg2+, ADP, ionic strength, and free energy of ATP hydrolysis by computation. 2) We will determine the influences these milieu changes have on: a) the Ca2+ uptake, and Ca2+ release in skinned fibers and SR triads, terminal cisternae, and longitudinal SR, b) the contractile apparatus by measuring the contractile properties of skinned single fibers. the studies utilizing skinned fibers will be carried out on preparations from control and fatigued muscles. These experiments will provide a better understanding of the cellular basis of fatigue than previous studies because this complex problem can only be successfully addressed with a multifaceted approach which incorporates both biochemical and physiological techniques that are applied to the same experimental model under the same experimental conditions. They will also determine whether the deficiencies with fatigue are the result of defects in the organelles themselves or whether it is the intracellular milieu that interferes with the normal function of the cell components.