Acetyl-CoA carboxylase (EC 6.4.1.2) activity in the supernatant fraction of the rat brain is highest during the first 2 weeks of postnatal life, coincident with the period of maximal brain growth, and then declines by 50% to adult levels. As its in vitro activity is similar to that of fatty acid synthetase, it does not appear to be the rate-limiting step in de novo fatty acid synthesis in brain. Both acetyl-CoA carboxylase and fatty acid synthetase activities are significantly inhibited by palmityl-CoA. Comparison of the Km (acetyl-CoA) of newborn and adult rat brain supernatant fractions reveals a fourfold difference, but this is probably not significant enough to indicate the presence of a newborn isoenzyme.