Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-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 epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-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(beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)