By examining the mechanical properties of single unactivated myofibrils it has been shown that shortening and stretching of sarcomeres occurs in stepwise fashion, and that steps are seen also in the relaxed state (Yang et al. (1998) Biophys J 74: 1473-1483; Blyakhman et al. (2001) Biophys J 81: 1093-1100; Nagornyak et al. (2004) J. Muscle. Res. Cell Motil. 25: 37-43). The latter are inevitably associated with connecting filaments. Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Myofibrils were stretched and released in ramp-like fashion. For the single sarcomere the time course of length change was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.