1. The mechanical signal(s) that may be important in regulating the number of sarcomeres in series in skeletal muscle fibres or fascicles (sarcomere number) remain(s) speculative, in part because the in vivo mechanical environment of muscle has not yet been defined during sarcomere number perturbations. In the present study, measurements of the in vivo mechanical environment were used to test the hypothesis that increasing muscle excursion results in increased serial sarcomere addition in growing animals. 2. The tibialis anterior (TA) was released from its retinacular restraint at the ankle joint to increase muscle excursion in 4-week-old female New Zealand White rabbits. Twelve weeks post release, muscle excursion and sarcomere number were significantly increased for released TAs compared with control TAs. 3. General cage activity over a 24 h period and in vivo ankle joint kinematics of the experimental leg during hopping on a treadmill were not significantly different between control and release groups, suggesting that altered animal and joint activity patterns were not responsible for the increased serial sarcomere addition associated with TA release. 4. In vivo TA forces during hopping and during a large force-producing foot-flicking motion were significantly decreased in released TAs compared with control TAs. Chronically decreased force production may have been involved in the decreased longitudinal tendon growth observed for the released TA compared with the control TA, which, in turn, may have stimulated the increase in serial sarcomere addition. However, increasing force production of the released TA by partial ablation of the extensor digitorum longus did not inhibit the increase in serial sarcomere addition. 5. The results of this study support the hypothesis that increasing excursion results in increased serial sarcomere addition in growing animals and, when combined with the results of previous studies indicating that decreasing excursion results in decreased serial sarcomere addition, support the working hypothesis that excursion is important in regulating sarcomere number in growing animals.