We studied the effects of (post-tetanic) potentiation on myosin light chain (MLC-2) phosphorylation, work and energy cost in skeletal muscle. Experiments were performed using in situ medial gastrocnemius muscles of male Wistar rats, which were electrically stimulated through the severed sciatic nerve. One group of muscles was first potentiated with an isometric tetanus before a series of 10 concentric contractions (PRC). A second group performed the same series of contractions without previous potentiation (RC). Following the last contraction the muscles were rapidly frozen and excised after which the high-energy phosphate content, lactate concentration and the level of MLC-2 phosphorylation were measured. The results indicate that PRC muscles had a higher (P < 0.05) total work output 144.5 ± 17.0 (SD) (n = 6) vs. 121.6 ± 11.4 (SD) (n = 6) mJ and level of MLC-2 phosphorylation (49.2 ± 7.3 vs. 40.8 ± 3.6%) than RC muscles. The energy cost of the series of concentric contractions in the PRC muscles (9.8 ± 1.9 μmol∼P/muscle) was significantly higher (P < 0.05) than the energy cost in the RC muscles (6.2 ± 0.97 μmol∼P/muscle). It was shown that the relative increase in energy cost of PRC muscles was higher (P < 0.05) than in total work output. It is proposed that the relative high increase in energy cost is the direct result of the increase in muscle performance rather than a property of potentiation.