The fluorescent analog of ATP 1-N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) exchanges readily with nucleotides bound to G-actin. The exchange can be observed by measuring the fluorescence intensity, which increases significantly when epsilon-ATP binds to actin. When excess epsilon-ATP is added to a solution of F-actin, a continuous increase in fluorescence intensity is observed, indicating that the nucleotides bound to F-actin are directly or indirectly exchangeable. The kinetics of exchange consist of a fast phase and a slow phase. Both phases are stimulated by shearing and are inhibited by phalloidin treatment, suggesting that the exchange of nucleotides is coupled to the exchange of subunits. Therefore, the exchange reaction can be used as a convenient, nonperturbing tool to study the exchange of free actin subunits with subunits in actin filaments. The exchange of actin subunits was characterized by a pulse-chase experiment. The results suggest that actin subunits assemble and disassemble through the same end of the filament during the fast phase of exchange but through opposite ends of the filament during the slow phase. In addition, the slow phase of exchange is inhibited in the absence of millimolar magnesium ions, but is not significantly affected by cytochalasin B at concentrations between 0.1 and 10 microM. These observations are discussed in relation to possible mechanisms of subunit exchange in steady-state F-actin solutions.