An acetyl‐CoA carboxylase has been purified from rat hindlimb muscle using ammonium sulfate fractionation and avidin‐Sepharose affinity chromatography. SDS/PAGE of the isolated enzyme showed a major protein band at approximately 272 kDa and a minor band at 265 kDa. The liver acetyl‐CoA carboxylase gave a major protein band at 265 kDa and a minor band at 280 kDa. Adipose tissue acetyl‐CoA carboxylase migrated to the 265‐kDa position on the gel. Western blots performed using streptavidin–alkaline‐phosphatase suggest that the bands from the three tissues contain biotin. The present study has characterized the muscle and adipose tissue enzymes under steady‐state kinetics and determined Michaelis constants for the substrates. The activation constant for citrate, an essential activator for both preparations, was 2.13 ± 0.05 mM for the muscle enzyme and 3.02 ± 0.12 mM for adipose tissue (P < 0.01). The K_m values for the muscle acetyl‐CoA carboxylase compared to the adipose tissue acetyl‐CoA carboxylase were: ATP, 57.6 ± 0.9 μM compared to 106.5 ± 2.6 μM, P < 0.01; acetyl‐CoA, 31.7 ± 1.5 μM compared to 21.5 ± 1.0 μM, P < 0.01; bicarbonate, 2.25 ± 0.10 mM compared to 2.73 ± 0.29 mM, P > 0.05. The muscle acetyl‐CoA carboxylase was inhibited by malonyl‐CoA (K_i= 10.6 ± 1.0 μM) and palmitoyl‐CoA (K_i= 2.2 ± 0.3 μM). These properties are consistent with the hypothesis that regulation of acetyl‐CoA carboxylase plays an important role in governing the rate of fatty acid oxidation in the skeletal muscle.