Summary A soluble protein that binds malonyl-CoA without requiring cofactors has been purified from rat liver. Until saturated, it competes with fatty acid synthetase for free malonyl-CoA, temporarily reducing the rate of fatty acid synthesis at low levels of malonyl-CoA, as in fatty acid synthetase—coupled assays for acetyl-CoA carboxylase. These assays yield low estimates for carboxylase activity with crude and partially purified homogenates containing the malonyl-CoA—binding protein. The protein does not inhibit assays for carboxylase activity that measure nonvolatile radioactivity incorporated from bicarbonate or NADH oxidation coupled to ADP formation. It has an M r of 180,000 and a subunit of 90,000. It has a lower affinity for ATP, ADP, and acetyl-CoA and none for CO 2 or fatty acid synthetase. No enzymatic function has been identified. The protein may regulate malonyl-CoA—binding enzymes.