The carnitine acetyltransferase and glutamate dehydrogenase activities of guinea-pig liver and other tissues were estimated. Both enzymes are wholly mitochondrial, and can only be fully observed after disruption of the mitochondrion. Triton X-100 (0·1%) or freeze-drying revealed more activity than other methods tried. In mitochondria prepared and suspended in 0·25 m -sucrose and in cell cytoplasm only small fractions of the total enzymic activity could be observed in guinea-pig liver: on average 7·5% of carnitine acetyltransferase and 5·5% of glutamate dehydrogenase. It is concluded that, in liver or mammary gland of goat, guinea pig or rat, little or no carnitine acetyltransferase is available in vivo to acetyl-CoA outside the mitochondrion.