Work output of rat gastrocnemius medialis (GM) muscle (N = 5) was measured for stretch-shortening contractions, in which initiation of stretch occurred prior to the onset of activation, and for contractions with an isometric prephase. Duration of the active prephase (prestretch and pre-isometric) varied from 20 to 200 ms. Subsequent shortening (from optimum length + 4 mm to optimum length -2mm) lasted 150 ms. Stretch velocities of 5, 10 and 20 mm s-1 were used, and the shortening velocity was 40 mm s-1. The effects of several combinations of active stretch duration and active stretch amplitude were compared. Using force-compliance characteristics, the work of the contractile element (CE), elastic energy storage and release of the undamped series elastic component (SEC) were distinguished. During shortening, an extra amount of work output was produced, induced by active stretch, of which the largest contribution (70-80%) was due to higher elastic energy release. Enhancement of the storage and utilization of elastic energy during the stretch-shortening cycle, caused by higher transition-point forces (i.e. force at onset of shortening), increased with active stretch amplitude and was associated with a net loss of work, probably due to cross-bridge detachment during active stretch. Net work over the stretch-shortening cycle remained positive for all prestretch contractions, indicating that when a muscle performs this type of contraction, it is able to contribute to work performance on body segments. It is concluded that, in stretch-shortening movements of rat GM muscle, maximal positive work output is incompatible with maximal net work output. Consequences for complex movements in vivo are discussed.