Contraction of skeletal muscle generates pressure stimuli to intramuscular tissues. However, the effects of pressure stimuli, other than those created by electricity or nerve impulse, on physiological and biochemical responses in skeletal muscles are unknown. The purpose of this study is to examine the effects of a pure pressure stimulus on metabolic responses in a skeletal muscle cell line. Atmospheric pressure was applied to L6 myoblasts using an original apparatus. Succinate dehydrogenase (SDH) activity was evaluated by colorimetric assay using tetrazolium monosodium salt. The amounts of 2-[³H]-deoxy-glucose uptake and lactate release were measured. SDH activity was 2.6- to 2.9-fold higher in pressurized L6 cells than in non-pressurized L6 cells (P < 0.01), and 2-[³H]-deoxy-glucose uptake was 2.2-fold higher (P < 0.001). In addition, the amount of released lactate decreased from 6.8 to 3.7 µmol/dish when pressure was applied (P < 0.001). In contrast, the intracellular lactate contents of the pressurized cells were higher than of non-pressurized cells (P < 0.01). However, the total amount of released lactate and intracellular lactate was lower in the pressurized cells than in non-pressurized cells. These findings demonstrate that a pure pressure stimulus enhances aerobic metabolism in L6 skeletal muscle cells, and raise the possibility that elevated intramuscular pressure during muscle activity may be an important factor in stimulating oxidative metabolic responses in skeletal muscles.