In attempting to deduce the size of the elementary molecular translation step, recent experiments using single myosin molecules translating over actin filaments have shown a consistent step size of 5.4 nm (10, 21). We have carried out parallel measurements on single myofibrils from rabbit cardiac muscle and bumblebee flight muscle. Activated specimens were released or stretched with a motor-imposed ramp, and the time course of length of individual sarcomeres was measured by projecting the image of the striations onto a linear photodiode array and tracking the spacing between A-band centroids. We confirmed the 5.4-nm step. With subnanometer precision, however, we find that this value is two times that of a more fundamental step size of 2.7 nm. Step sizes were always integer multiples of 2.7 nm, whether the length change was positive or negative. This value is equal to the linear repeat of actin monomers along the thin filament, a result that ties dynamic events to molecular structure and places narrow constraints on any proposed molecular mechanism.