Mitochondria do not have the enzyme, methionine adenosyltransferase (ATP: L-methionine S-adenosyltransferase, EC 22.214.171.124), necessary for the biosynthesis of S-adenosylmethionine. Nevertheless, about 30% of total hepatic S-adenosylmethionine resides in the mitochondria and radiolabeled S-adenosylmethionine may be isolated from the mitochondria after administration of radiolabeled methionine. This leads to the hypothesis that a carrier-mediated system is responsible for S-adenosylmethionine transport from the cytosol into the mitochondria. We have characterized such a system in isolated rat liver mitochondria. Uptake of S-adenosylmethionine consisted of two components. One component was incorporation of the methyl group into phospholipids as shown by thin-layer chromatography. The second component represented uptake into the mitochondria since addition of excess unlabeled S-adenosylmethionine resulted in efflux of labeled substrate. This countertransport is characteristic of a carrier-mediated transport system. Uptake (corrected for incorporation into phospholipids) was saturable with an apparent Km = 8.9 microM and Vmax = 54.3 pmol x mg protein(-1) x min(-1). Uptake was not inhibited by methionine, adenosine, 5'-methylthioadenosine, carnitine, choline, betaine, quinine, or hemicholinium-3. Uptake was inhibited by sinefungin and by S-adenosylhomocysteine (Ki = 53.4 microM). Uptake of S-adenosylmethionine was not dependent on the electrical potential across the mitochondrial membrane. These results indicate that S-adenosylmethionine is taken up into mitochondria via a specific, carrier-mediated system.