The polymerization of rabbit skeletal muscle actin was studied with the technique of fluorescence photobleaching recovery by using trace amounts (5%) of actin labeled with (iodoacetamido)tetramethylrhodamine as the fluorescent probe. This derivative's fluorescence increases 50% upon polymerization, so the rate and extent of polymer formation can be measured from the same sample used in the photobleaching experiment. When photobleaching experiments are done under conditions suitable for following the diffusion of polymeric actin, the progress of polymerization at 1 mg/mL and 2 mM Mg2+ is as follows: (1) Prior to significant accumulation of polymer, all the actin diffuses too rapidly to be observed. (2) As polymer forms, it diffuses with an apparent diffusion coefficient in the range of (~0.05-0.5) X 10-8 cm2/s. (3) Before completion of polymerization, filaments become immobilized (apparent diffusion coefficient <<2 X 10-11 cm2/s). The second stage probably represents growing polymers whose diffusion is hindered by interaction with other polymers; the third stage is attributed to immobilization of filaments in networks. Immobilization can be reversed by shearing the sample, but the filaments become immobile again within 3-5 min under these conditions. Immobilization occurs at a lower concentration of F-actin when the total actin concentration is reduced at constant Mg2+ concentration or when actin is polymerized with KC1 rather than Mg2+. These results suggest that filaments are longer under conditions where the rate of polymerization is slow. Polymerization in the presence of cytochalasin D (2µM) delays the onset of immobilization until after a steady-state concentration of F-actin is reached, indicating that filaments grown in the presence of cytochalasin are initially short but gradually lengthen. Polymerized actin (1 mg/mL, 2 mM Mg2+) almost completely prevents the diffusion of 250-nm fluorescent beads, suggesting that the effective pore size of the actin network is less than 250 nm. We conclude that fluorescence photobleaching recovery experiments can measure relative sizes of actin filaments, filament annealing, and network formation.