Lysosome-free preparations of rat liver mitochondria take up hydroxo[57Co]cobalamin by a process which is dependent on mitochondrial swelling, rather than on energy or ion fluxes. The uptake system is saturable and unidirectional, leading to inside/outside concentration ratios of 17. The process also exhibits specificity: cyano[57Co]cobalamin is taken up less rapidly and to a lesser extent than hydroxocobalamin; methylcobalamin and adenoslcobalamin inhibit hydroxocobalamin uptake markedly, while cyanocobalamin does not. The [57Co]cobalamin ([57Co]Cbl) taken up is bound to a mitochondrial protein whose apparent molecular weight is 120,000 by Sephadex G-150 chromatography. Double reciprocal plots of bound [57Co]Cbl versus medium [57Co]Cbl concentration yield estimates for bound Cblmax of 29 pmol/mg of protein and for Kd is 8.2 muM. We conclude that mitochondrial uptake of cobalamins occurs via the diffusion of free cobalamins into the mitochondria and their subsequent binding to a high affinity mitochondrial protein(s) which we propose to be the source of the unidirectional character, the saturability, and the specificity of the uptake system.