Rabbit skeletal muscle G-actin has been treated to obtain ADP, l, N6-ethenoadenosine diphosphate (є-ADP), or 1, N6-ethenoadenosine triphosphate (є-ATP) at the nucleotide binding site and either Mg2+or Ca2+at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by є-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 °C of the fluorescence difference between bound and free ∊-ATP or ∊-ADP. In the presence of Ca2+, displacement of ADP by є-ATP or є-ADP by ATP is a biphasic process, but in the presence of low (<10 μM) Mg2+concentrations, it is a slow first-order process. At high levels of Mg2+(>50 μM), low ADP concentrations displace є-ATP from G-actin as a consequence of Mg2+binding to moderate-affinity sites on the actin. Displacement of є-ATP by ATP in the presence of either Ca2+or Mg2+is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+from the high-affinity site compared to Ca2+removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP. It is concluded that, in general, the presence of Ca2+strengthens ATP binding relative to ADP, while the presence of Mg2+weakens ATP binding relative to ADP.