Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.