Tubercidin nucleotides [tubercidin 5'-mono-phosphate (TuMP), 5'-diphosphate (TuDP), and 5'-triphosphate (TuTP)] were tested as potential substrates for the mitochondrial phosphotransferases from rat liver and beef heart. TuDP is recognized by the mitochondrial translocase and phosphorylated by the respiratory chain enzymes in both mitochondria and submitochondrial particles from rat liver and beef heart; the low transport rate of the analogue into the matrix space of the intact organelles seems to be not a limiting step in the formation of TuTP. The phosphorylation of TuDP is significantly lower in beef heart mitochondria because of a higher specificity for ADP of the heart oxidative phosphorylation system. On the basis of the kinetic parameters of the partially purified liver mitochondrial adenylate kinase, one can conclude that the liver mitochondria are able to phosphorylate in vivo TuMP at a rate practically equal to the rate of AMP phosphorylation. The liver mitochondrial NDP kinase ensures a further fast phosphorylation of TuDP without the direct involvement of respiratory chain enzymes. In the case of heart mitochondria, two factors limit the rate of TuMP phosphorylation to TuTP: the lower acceptor activity of adenylate kinase with TuMP as compared with AMP and the different localization of heart NDP kinase situated on the inner face of the inner mitochondrial membrane. TuDP and TuTP preserve the ability of the natural nucleotides to interact with the "tight" nucleotide binding sites of isolated or membrane-bound F1. The low hydrolytic rate of TuTP with F1 may be related to the unusual flexibility of the glycosyl bond of tubercidin nucleotides in aqueous solution, with a high accessibility to syn conformation.