We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodoacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enhanced up to 50% by profilin. The higher fluorescence is largely due to higher absorbance in the presence of profilin. The fluorescence enhancement has a hyperbolic dependence on the concentration of profilin, suggesting a single class of binding sites. Linear Scatchard plots yield an estimate of the dissociation constant, Kd, of the complex of profilin with pyrenyl-actin. In low-ionic-strength buffers with 2 to 6 mM imidazole (pH 7.0) and 0.1 mM CaCl2 the Kd is 9 microM for both muscle and Acanthamoeba actin. In 50 mM KCl the Kd for the complex with Acanthamoeba actin is 16 microM, while the Kd for the complex with muscle actin is greater than 50 microM.