Flux through anaplerotic pathways in skeletal muscle has not been evaluated quantitatively during both rest and contraction, nor have fibre type-specific rates of anaplerotic flux been studied. Steady-state analysis using ¹³C NMR spectroscopy enables calculation of Y (flux rate through anaplerotic pathways relative to tricarboxylic acid (TCA) cycle flux). Under inhalation anaesthesia, [2,4,6,8-¹³C₄]octanoate was infused into the jugular vein of the intact rat (n = 10) and the sciatic nerve of one limb was stimulated at the voltage required to elicit maximal force output at 0.5 Hz. In resting muscle, Y was higher in soleus (0.41 ± 0.22) versus white gastrocnemius (WG) (0.18 ± 0.11). Y was 0.29 ± 0.06 in the predominantly red portion of the gastrocnemius (RG) during rest. During contraction, Y was similar to the resting value in soleus (0.34 ± 0.14), RG (0.20 ± 0.04) and WG (0.15 ± 0.08); Y was higher in soleus versus both RG and WG during contraction. These results demonstrate: (1) relative flux through anaplerotic pathways is 15-41 % of TCA cycle flux at rest and during muscle contraction, (2) higher relative anaplerotic flux in oxidative (soleus) versus glycolytic muscle (WG) during rest and in slow-twitch (soleus) versus fast-twitch (RG and WG) muscle during contraction, and (3) relative flux through anaplerotic pathways is maintained in all muscle fibre types during contraction, which indicates that absolute rates of anaplerotic flux rise in proportion to increased oxidation rates during contraction. These results are consistent with a sustained increase in substrate entry into and exit from the TCA cycle through anaplerotic pathways during contraction.