The phosphorylation of various intact tRNA species by T4 polynucleotide kinase has been studied. The apparent Michaelis constant was on the average found to be 100 times lower than for some single-stranded DNAs previously studied. (J.R. Lillehaug and K. Kleppe, (1975) Biochemistry, 14, 1221). Conditions which result in complete phosphorylation of different tRNA species have also been established. Studies on equilibrium constants and the reversibility of the reaction revealed that the phosphorylation reaction is not a true equilibrium reaction under the conditions used in this work.