The functional characteristics of the various types of skeletal muscles, are largely dictated by the expression of class II myosin heavy chains (MyHC) isoforms; especially contraction velocity, a manifestation of the rate of actomyosin (A.M.) interaction and of ATP turnover. Higher levels of expression of a fast MyHC isoform lead to faster contraction velocity, while higher levels of expression of a slow MyHC lead to a slower contraction velocity. It has been observed that in slow myosin isoforms ADP binding to A.M is tighter than with fast isoforms, and one of us has shown that with slow isoforms multiple A.M.ADP complexes are present. The in vivo muscle temperature ranges from 34 to > 40 degrees Celsius (oC) while in extreme fatigue, pH drops and inorganic phosphate (Pi) accumulates. Still, most kinetics characterisations in the past have been done in ‘standard’ conditions e.g. 20oC, pH 7 and no added phosphate. Recent data by one of us highlighted the importance of investigating possible synergisms between temperature and such ‘fatigue’ factors which may gain different ‘importance’ in the different fibre types. We thus examined the fast kinetics of ATP induced dissociation of A.M. (with and without ADP) of rabbit fast and slow myosin for a range of temperatures (from 5 to 35oC) using stopped flow to examine the effects of pH (7 vs 6.2), and Pi (0, 15 and 30 mM), individually or in combination. We also examined the temperature dependence of these ‘fatigue’ factors. The experimental protocol was approved by the local Animal Ethics Committee, and all the experimental procedures conformed to the UK Animals (Scientific Procedures) Act, 1986. Temperature did not have an evident effect on ADP affinity nor did low pH seem to affect ADP affinity for the fast myosin. For slow myosin, ADP affinity weakened at pH 6.2, also at 35oC. Addition of Pi affected the two myosin typess differently, Pi competing with the ADP inhibition in fast but almost abolishing ADP inhibition in slow myosin at the higher Pi concentration. In slow myosin Pi and low pH combined affected ATP induced dissociation of A.M in the lower range of the temperatures studied. Our results provide further evidence of distinct mechanochemical coupling between the myosin types. Results will be also discussed in relation to the effects of fatigue on velocity of contraction.