Cytoplasmic actin from cultured fibroblasts has been purified to homogeneity and characterized with respect to its polymerization and structure. It was found to be qualitatively similar to muscle actin in all respects, but significant quantitative differences in its properties were demonstrated. Although BHK actin did not polymerize in unfractionated cytoplasmic extracts, the purified BHK actin polymerized into filaments both in magnesium and calcium. The critical concentration, measured by the DNase I inhibition assay and by fluorimetry, was the same as that of muscle actin both in magnesium and calcium. Polymerization of pyrene-labelled BHK and muscle actin was followed by fluorimetry. Significant differences in kinetics were found under both ionic conditions tested. In the absence of Mg2+ ions (0.2 mM CaCl2, 85 mM KCl), BHK actin polymerized at a much slower rate than muscle actin. In the presence of magnesium and EGTA, the nucleation phase for BHK actin polymerization was shorter than that for muscle actin and the kinetics of polymerization was different. The structure of BHK actin filaments in the electron micrographs was very similar to that of muscle actin. In high concentrations of magnesium, BHK actin formed paracrystals which had the same appearance as muscle actin paracrystals. However, calcium-induced formation of actin paracrystals required higher concentration of Ca2+ ions for BHK actin than for muscle actin (12 mM and 8 mM respectively). These results suggest differences in divalent cation binding to both high- and low-affinity sites of the two actins.