The transport of radioactive glycine, serine, and proline into the matrix of spinach leaf mitochondria was studied using the silicone oil centrifugation technique. The uptake of all three amino acids showed a biphasic characteristic. At concentrations higher than 0.5 mM, an apparent diffusion process dominated. The uptake was not saturable at increasing amino acid concentrations, and there was no accumulation of amino acid in the matrix (i.e., concentration was similar to that in the medium). At concentrations lower than 0.5 mM, in addition to the diffusion process, an active uptake system that accumulated amino acid in the matrix became apparent. This system was partially inhibited by rotenone, antimycin A, and carbonylcyanide-m-chlorophenyl hydrazone. Also, uptake of glycine and serine was mutually inhibitory. These two amino acids exhibited comparatively less inhibitory effect on proline uptake, and proline also did not inhibit glycine or serine uptake. The results suggest that the active uptake system consists of at least two components with different degrees of amino acid specificity. The diffusion process dominates at amino acid concentrations of 0.5 mM or higher, whereas the active uptake system becomes more prominent as the amino acid concentration decreases.