The effect of prior eccentric contractions on skeletal muscle lactate/H⁺ transport was investigated in rats. Lactate transport was measured in sarcolemmal giant vesicles obtained from soleus and red (RG) and white gastrocnemii (WG) muscles 2 days after intense eccentric contractions (ECC) and from the corresponding contralateral control (CON) muscles. The physiochemical buffer capacity was determined in the three muscle types from both ECC and CON legs. Furthermore, the effect of prior eccentric contractions on release and muscle content of lactate and H⁺ during and after supramaximal stimulation was examined using the perfused rat hindlimb preparation. The lactate transport rate was lower (P < 0.05) in vesicles obtained from ECC-WG (29%) and ECC-RG (13%) than in vesicles from the CON muscles. The physiochemical buffer capacity was reduced (P < 0.05) in ECC-WG (13%) and ECC-RG (9%) compared with the corresponding CON muscles. There were only marginal effects on the soleus muscle. Muscle lactate concentrations and release of lactate during recovery from intense isometric contractions were lower (P < 0.05) in ECC than in CON hindlimbs, indicating decreased anaerobic glycogenolysis. In conclusion, the sarcolemmal lactate/H⁺ transport capacity and the physiochemical buffer capacity were reduced in prior eccentrically stimulated WG and RG in rats, suggesting that muscle pH regulation may be impaired after unaccustomed eccentric exercise. In addition, the data indicate that the glycogenolytic potential is decreased in muscles exposed to prior eccentric contractions.