Recent developments of in vitro motility assays have allowed the sliding velocity and force generation to be measured when a single actin filament interacts with a small number of immobilized myosin molecules. In contrast, the associated ATPase activities have been estimated from the whole population of molecules in the flow cell using steady-state kinetics. For a more unambiguous estimate of the crossbridge step size, it would be desirable to measure the ATPase activity of those molecules actually under observation in the in vitro assay in real time. As a start to solving this formidable problem we have investigated the use of fluorescent ATP analogues as probes to measure the ATPase activity of immobilized myosin filaments. Turnover rates for the substrate analogue, FEDA-ATP (an analogue in which a fluorescein moiety is linked via an ethylenediamine chain to the ribose of ATP) were recorded by displacement of the steady-state intermediate with excess ATP. Using epifluorescence light microscopy, small clumps of rabbit skeletal and scallop striated muscle synthetic thick filaments and single native clam ( Mercenaria) red adductor muscle thick filaments were assayed. The latter filaments contain several thousand myosin molecules and thus they represent the application of transient kinetic methodology on the zeptomole scale. In the presence of Ca 2+, the derived rate constants for FEDA-ATP turnover are close to those expected for the same preparations in solution (0·06 s -1 for rabbit skeletal, 0·2 s -1 for scallop and clam adductor muscle myosins), indicating that immobilization does not have a significant effect on the ATPase activity. In the absence of Ca 2+, molluscan preparations show a slower FEDA-ATP turnover rate but they do not appear as well regulated as in solution. In such microscope assays the observed displacement rate does not reflect the true turnover rate owing to photobleaching, and possibly regulatory light chain depletion. Future developments for extending this assay to the actin-activated state are discussed.