When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20 degrees C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.