Metabolism-dependent inactivators of 3-ketothiolase I and carnitine acyltransferase I (CAT I) have been used to study the oxidation of fatty acids in intact hepatocytes. 2-Bromooctanoate inactivates mitochondrial and peroxisomal 3-ketothiolases I in a time-dependent manner. During the first 5 min of incubation, inactivation of 3-ketothiolase in mitochondria is five times faster than its inactivation in peroxisomes. Almost complete inactivation of 3-ketothiolase I in both types of organelle is achieved after incubation with 1 mM 2-bromooctanoate for 40 min. The inactivation is not affected by preincubating hepatocytes with 20 microM tetradecylglycidate (TDGA), an inactivator of CAT I, under conditions which cause greater than 95% inactivation of CAT I. 2-Bromododecanoate (1 mM) causes 60% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min. These inactivations are greatly reduced by preincubating hepatocytes with 20 microM TDGA, demonstrating that 2-bromododecanoate enters both mitochondria and peroxisomes via its carnitine ester. 2-Bromopalmitate (1 mM) causes less than 5% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min, but causes 95% inactivation of CAT I during this time. Incubation of hepatocytes with 10-200 microM 2-bromopalmitoyl-L-carnitine causes inactivation of mitochondrial and peroxisomal 3-ketothiolases I at similar rates. This inactivation is decreased by palmitoyl-D-carnitine during the first 5 min of incubation. Pretreating hepatocytes with 20 microM TDGA does not affect the inactivation of mitochondrial or peroxisomal 3-ketothiolase I by 2-bromopalmitoyl-L-carnitine. These results demonstrate that in intact hepatocytes, peroxisomes oxidize fatty acids of medium-chain length by a carnitine-independent mechanism, whereas they oxidize long-chain fatty acids by a carnitine-dependent mechanism.