The kinetics of L-[14C]arginine (L-[14C]Arg) uptake and the effects of potential competitors on the uptake were analysed in nonsynaptic mitochondria isolated from rat cerebral hemispheres. Analysis of uptake kinetics revealed a high affinity component with a mean Km = 0.08 mM, and Vmax = 1.89 nmoles/min/mg, and a very low affinity component probably manifesting diffusion. The uptake of 25 mM L-Arg was strongly inhibited by a 20-fold excess of L-lysine (L-Lys) and L-ornithine (L-Orn), but not by D-Arg nor any neutral amino acid, which resembles the characteristics of the gamma+ transport system operating in the nerve- and glia cell-, and synaptic plasma membranes. Also in consistance with the other gamma+ systems, L-Arg uptake to mitochondria was inhibited by a nitric oxide synthase (NOS) inhibitor L-N-monomethyl arginine (L-NMMA), but not by another NOS inhibitor NG-nitro-L-arginine (L-NNA). However, the uptake was very little affected by 20-fold excess of L-histidine (L-His), L-glutamate (L-Glu) or L-glutamine (L-Gln), which is in contrast to the nonmitochondrial systems. The uptake was only marginally influenced by cytoplasmic L-Arg metabolites: ammonia, creatine, putrescine, or the mitochondrial L-Arg decarboxylation product, agmatine.