Abstract: The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20‐ and 60‐day‐old rats were incubated with [³H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 μM), or rotenone (10 μM) reduced the incorporation of [³H]palmitate into fatty acyl‐CoA and glycerolipids by 50‐60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 μM) plus NaF (5 mM) abolished the synthesis of acyl‐CoA and lipid palmitoylation, but the incorporation of [³H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP‐dependent and ATP‐independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP‐dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP‐independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.