We examined length changes of individual half-sarcomeres during and after stretch in actively contracting, single rabbit psoas myofibrils containing 10-30 sarcomeres. The myofibrils were fluorescently immunostained so that both Z-lines and M-bands of sarcomeres could be monitored by video microscopy simultaneously with the force measurement. Half-sarcomere lengths were determined by processing of video images and tracking the fluorescent Z-line and M-band signals. Upon Ca2+ activation, during the rise in force, active half-sarcomeres predominantly shorten but to different extents so that an active myofibril consists of half-sarcomeres of different lengths and thus asymmetric sarcomeres, i.e. shifted A-bands, indicating different amounts of filament overlap in the two halves. When force reached a plateau, the myofibril was stretched by 15-20% resting length (L0) at a velocity of approximately 0.2 L0 s(-1). The myofibril force response to a ramp stretch is similar to that reported from muscle fibres. Despite the approximately 2.5-fold increase in force due to the stretch, the variability in half-sarcomere length remained almost constant during the stretch and A-band shifts did not progress further, independent of whether half-sarcomeres shortened or lengthened during the initial Ca2+ activation. Moreover, albeit half-sarcomeres lengthened to different extents during a stretch, rapid elongation of individual sarcomeres beyond filament overlap ('popping') was not observed. Thus, in contrast to predictions of the 'popping sarcomere' hypothesis, a stretch rather stabilizes the uniformity of half-sarcomere lengths and sarcomere symmetry. In general, the half-sarcomere length changes (dynamics) before and after stretch were slow and the dynamics after stretch were not readily predictable on the basis of the steady-state force-sarcomere length relation.