We analyzed the distribution of actin filament lengths by optical microscopy (OM). OM avoids possible alterations in the size or structure of actin filaments occurring during sample preparation for electron microscopy (EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were analyzed for both size distribution and flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4, 25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM beta-mercaptoethanol] filaments did not aggregate into bundles and remained stable at nanomolar concentrations for at least 1 h. At the same concentration, actin labeled directly with rhodamine (no phalloidin) formed unstable filaments whose average length decreased with time. The number average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns, the ratio (rho) of the weight average length to the number average length was 2.06, and the correlation length (1/lambda) was 8.33 microns. These parameters were in good agreement with the values determined by EM for filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150 column before polymerization did not have a significant effect on the distribution of lengths but made filaments more stiff (1/lambda = 12.5 microns). Millimolar concentration of ATP increased the correlation length, and gelsolin had the expected fragmenting effect on filaments. These results show that OM can be used as a fast and reliable method to analyze the distribution and flexibility of actin filaments and suggest that, in spite of extensive manipulation of actin filaments during sample preparation, EM is a valid tool for determination of size parameters of actin filaments.