Abstract: The depression in force and/or velocity associated with muscular fatigue can be the result of a failure at any level, from the initial events in the motor cortex of the brain to the formation of an actomyosin cross-bridge in the muscle cell. Since all the force and motion generated by muscle ultimately derives from the cyclical interaction of actin and myosin, researchers have focused heavily on the impact of the accumulation of intracellular metabolites [e.g., P(i), H(+) and adenosine diphoshphate (ADP)] on the function these contractile proteins. At saturating Ca(++) levels, elevated P(i) appears to be the primary cause for the loss in maximal isometric force, while increased [H(+)] and possibly ADP act to slow unloaded shortening velocity in single muscle fibers, suggesting a causative role in muscular fatigue. However the precise mechanisms through which these metabolites might affect the individual function of the contractile proteins remain unclear because intact muscle is a highly complex structure. To simplify problem isolated actin and myosin have been studied in the in vitro motility assay and more recently the single molecule laser trap assay with the findings showing that both P(i) and H(+) alter single actomyosin function in unique ways. In addition to these new insights, we are also gaining important information about the roles played by the muscle regulatory proteins troponin (Tn) and tropomyosin (Tm) in the fatigue process. In vitro studies, suggest that both the acidosis and elevated levels of P(i) can inhibit velocity and force at sub-saturating levels of Ca(++) in the presence of Tn and Tm and that this inhibition can be greater than that observed in the absence of regulation. To understand the molecular basis of the role of regulatory proteins in the fatigue process researchers are taking advantage of modern molecular biological techniques to manipulate the structure and function of Tn/Tm. These efforts are beginning to reveal the relevant structures and how their functions might be altered during fatigue. Thus, it is a very exciting time to study muscle fatigue because the technological advances occurring in the fields of biophysics and molecular biology are providing researchers with the ability to directly test long held hypotheses and consequently reshaping our understanding of this age-old question.