The fluorescence enhancement of actin labeled with N-(1-pyrenyl)iodoacetamide was used to monitor the rate of incorporation of monomeric G-actin into actin polymer. When such experiments are performed in undisturbed solutions, both the rate of polymerization and the extent of polymer formation can be quantitatively measured. Under all conditions, cytochalasin D accelerates polymerization as reflected by an enhanced rate of incorporation of monomer into polymer and/or a decreased polymerization lag time. However, the presence of cytochalasin D decreases the extent of polymer formation. The amount of polymer formed in the presence of cytochalasin D is more strongly dependent on the conditions under which polymerization occurs, i.e., the Mg2+ concentration and the temperature, than is the amount of polymer formed in the absence of cytochalasin D. A general observation is that in the presence of cytochalasin D, conditions that give slow rates of polymerization incorporate much less monomeric actin into polymer than conditions that give rapid rates of polymerization. The results are interpreted in terms of the nucleation-elongation model for actin polymerization and the supposition that cytochalasin binds preferentially to one end of a growing polymer. However, not all the observations may fit this simple model. We have isolated an actin binding protein from platelets that shows effects similar to those of cytochalasin D. This protein appears to correspond to macrophage gelsolin. It is proposed that preferential binding of such proteins to one end of a growing polymer may influence both the rate and extent of polymerization. If these parameters are altered by physiological conditions, certain actin binding proteins could regulate the concentration of free G-actin in vivo.