Author ContributionsA.M. and M.U. conceived the project, M.U. and A.M. designed the TIRF system. M.U. and A.M. designed the experiments. M.U. built the TIRF system and performed single molecule fluorescence experiments. M.U. L.M. and A.M. analyzed TIRF data, L.M. performed the bioinformatics modelling. V.V. performed in vitro motility assay experiments with and without AmBleb and analyzed the data, and A.S. analyzed in vitro motility assay data, A.M., M.U., L.M., V.V. and A.S. wrote the manuscript and approved the final version. AbstractSingle molecule enzymology using fluorescent substrate requires truly minimal amounts of proteins. This is highly beneficial when the protein source is either advanced expression systems or samples from humans/animals with ethical and economic implications. Further benefits of single molecule analysis is the potential to reveal phenomena hidden in ensemble studies. However, dye photophysics and fluorescent contaminants complicate interpretation of the single molecule data. We here corroborate the importance of such complexities using fluorescent Alexa647 ATP to study ATP turnover by myosin and actomyosin. We further show that the complexities are largely eliminated by aggressive surface cleaning and use of a range of triple state quenchers and redox agents with minor effects on actin-myosin function. Using optimized assay conditions, we then show that the distributions of ATP binding dwell times on myosin are best described by the sum of 2 to 3 exponential processes. This applies in the presence and absence of actin and in the presence and absence of the drug para-aminoblebbistatin. Two of the processes are attributable to ATP turnover by myosin and actomyosin, respectively. A remaining process with rate constant in the range 0.2-0.5 s -1 is consistent with non-specific ATP binding to myosin and bioinformatics modelling suggests that such binding may be important for accelerated ATP transport to the active site. Finally, we report studies of the actin-activated myosin ATP turnover under conditions with no sliding between actin and myosin, as in isometrically contracting muscle, revealing heterogeneity in the ATP turnover kinetics between different molecules.