The effects of high myoplasmic l-lactate concentrations (20–40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca2+ release, and passive Ca2+ leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. l-Lactate (20 mM) significantly reduced maximum Ca2+-activated force by 4 ± 0.5% ( n = 5, P < 0.05) and 5 ± 0.4% ( n = 6, P < 0.05) for EDL and soleus, respectively. The Ca2+ sensitivity was also significantly decreased by 0.06 ± 0.002 ( n = 5, P < 0.05) and 0.13 ± 0.01 ( n = 6, P < 0.001) pCa units, respectively. Exposure tol-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 ± 3% ( n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 μM), or the chloride channel blocker anthracene-9-carboxylic acid (100 μM). l-Lactate (20 mM) increased passive Ca2+ leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 ± 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 ± 2%, n = 3). These results indicate that the l-lactate ion per se has negligible effects on either voltage-dependent Ca2+ release or SR Ca2+ handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.