We have previously proposed that changes in malonyl-CoA sensitivity of rat liver carnitine palmitoyltransferase 1 (L-CPT1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we now prove experimentally the existence of such N/C intramolecular interactions both in wild-type L-CPT1 expressed in S. cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N/C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer-membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitisation, attenuated the N/C interactions. This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N/C interactions. Finally, we show that mutations in the regulatory regions of the N-domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased (S24AQ30A) or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to TM1 (residues 40-47) as being involved in the chemical N/C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.