AbstractThe interaction of mildronate [3‐(2,2,2‐trimethylhydrazine) propionate] with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Mildronate, externally added to the proteoliposomes, strongly inhibited the carnitine/carnitine antiport catalyzed by the reconstituted transporter with an IC50of 560 μM. A kinetic analysis revealed that the inhibition is completely competitive, that is, mildronate interacts with the substrate‐binding site. The half‐saturation constant of the transporter for external mildronate (Ki) is 530 μM. Carnitine/mildronate antiport has been measured as [3H]carnitine uptake into proteoliposomes containing internal mildronate or as [3H]carnitine efflux from proteoliposomes in the presence of external mildronate, indicating that mildronate is transported by the carnitine/acylcarnitine transporter and that the inhibition observed was due to the transport of mildronate in the place of carnitine. The intraliposomal half‐saturation constant for mildronate transport (Km) has been determined. Its value, 18 mM, is much higher than the external half‐saturation constant (Ki) in agreement with the asymmetric properties of the transporter. In vivo, the antiport reaction between cytosolic (administered) mildronate and matrix carnitine may cause intramitochondrial carnitine depletion. This effect, together with the inhibition of the physiological transport, will lead to impairment of fatty acid utilization. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:8–14, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20208