The binding of tropomyosin (Tm) to actin filaments was studied using the rotary shadowing technique. Electron micrographs showed, in addition to free Tm molecules, some Tm molecules bound to actin filaments at only one end creating ''tethered molecules.'' The ratio of free and bound Tm was determined by comparing densities of free Tm molecules in micrographs with those in Tm alone specimens. At a 1 to 7 ratio of 17 nM chicken gizzard Tm (gTm) and 120 nM actin filaments, the sum of the tethered (7%) and the free was only 29% of total Tm. The missing Tm molecules are likely to be those bound to actin filaments lengthwise and not visualized by the technique. At a lower ratio of Tm (3 nM) to actin (120 nM), the majority of bound Tm appeared to be tethered. At a lower actin concentration but a higher ratio of Tm (12 nM) to actin (24 nM), 57% of bound Tm appeared to be tethered. The populations of free and tethered molecules were also affected by different salt and glycerol concentrations or type of Tm used: more with rabbit skeletal (sTm) than with gTm. The tethering indicates that the interaction of Tm molecules to actin filaments is stronger at one end than at the rest of the molecule. The bundling of actin filaments was observed under certain conditions (e.g., similar to 2 mu M actin and similar to 0.5 mu M sTm), suggesting that both ends of Tm molecules can bind to actin filaments but one end binds much more weakly than the other. All of these observations support the fact that free and tethered molecules are under rapid equilibrium and that incidental encounter of the ends of two tethered Tm molecules which are at the right positions, or of one tethered and a free molecule, would strengthen the remaining actin binding site(s), resulting in tight binding, thus, the tethered molecules represent a weak form of binding. (C) 1996 Academic Press, Inc.