The fluorescence of pyrene-labelled actin is much higher after polymerization. We have characterized in detail the polymerization properties of pyrene actin and report that native and pyrene actin are identical using the following criteria: (1) the time course of polymerization; (2) the elongation rate constants; (3) the intrinsic viscosity; and (4) the critical concentration. Native and pyrene actin copolymerize. Fluorescence of polymerized pyrene actin is 7–10 times higher than monomer. The fluorescent signal is proportional to polymer weight concentration and is insensitive to filament length distribution. Bleaching can be minimized by appropriate filters to allow continuous monitoring of signal. Measurements do not influence polymerization kinetics. This establishes that pyrene actin fluorescence is a valid assay for actin polymerization that is more sensitive than any other current assay.