Studies of the interaction of proteolytic fragments of myosin with actin filaments in solution have indicated that the two heads of the myosin molecule1 can bind to adjacent subunits in the same actin filament2,3. However, geometrical considerations led Offer and Elliott4 to the conclusion that in the highly ordered filament lattice present in insect flight muscle, tighter binding would occur if the two heads bound to different actin filaments. Indeed, rigidity measurements 5, electron microscope observations6 and superprecipitation experiments7 support the idea that heavy meromyosin (HMM), the double-headed myosin fragment, can cross-link actin filaments in solution. We now provide an experimental demonstration of the cross-linking of thin filaments in muscle by double-headed myosin species. In striated muscle, the thin filament lattice can be stabilized by either the Z-bands or complex formation with myosin. Removal of the Z-bands by a proteolytic enzyme8 or extraction of myosin, leaves the actin-containing I-bands intact. However, if both Z-bands and myosin are removed, the I-bands should disintegrate into single, dispersed, filaments. We found that this could be prevented by preincubation with HMM, but the single -headed HMM subfragment-1 (HMM S-l) was totally ineffective. This strongly suggests that the stabilization by HMM of the filamentous lattice is a result of inter-filament cross-linking.