It is unclear whether accumulation of lactate in skeletal muscle fibers during intense activity contributes to muscle fatigue. Using mechanically skinned fibers from rat and toad muscle, we were able to examine the effect of l(+)-lactate on excitation-contraction coupling independently of other metabolic changes. We investigated the effects of lactate on the contractile apparatus, caffeine-induced Ca2+ release from the sarcoplasmic reticulum, and depolarization-induced Ca2+release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable (20–35%) inhibition of caffeine-induced Ca2+ release, seemingly by a direct effect on the Ca2+ release channels. However, 15 mM lactate had no detectable effect on Ca2+release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect on normal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.