It has been shown previously, using G-actin labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine, that Mg2+ induces a conformational change in monomeric G-actin as a consequence of binding to a tight divalent cation binding site (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Using the same fluorescent probe, we show that, subsequent to the Mg2+-induced conformational change, cytochalasin D induces a fluorescence decrease. The data are consistent with a mechanism which proposes that, after Mg2+ binding, cytochalasin D binds and induces a second conformational change which results in overall tight binding of the cytochalasin. The initial binding of cytochalasin D to monomeric actin labeled with the fluorescent probe was found to be 200 microM, and the forward and reverse rate constants for the subsequent conformational change were 350 s-1 and 8 s-1, respectively, with an overall dissociation constant to the Mg2+-induced form of 4.6 microM. The conformational change does not occur in monomeric actin in the presence of Ca2+ rather than Mg2+, but Ca2+ competes with Mg2+ for the tight binding site on the G-actin molecule. Direct binding studies show that actin which has not been labeled with the fluorophore binds cytochalasin D more tightly. The conformational change induced by Mg2+ and cytochalasin D precedes the formation of an actin dimer.