We have examined the cation requirements of rabbit liver tRNA nucleotidyltransferase. The enzyme had an absolute requirement fo a divalent cation which could be satisfied by Mg2+, Mn2+ or Co2+. In contrast to the Escherichia coli enzyme, we have found no evidence to implicate Zn2+ in the action of rabbit liver tRNA nucleotidyltransferase. We have also identified a second cation requirement which was satisfied by divalent or monovalent cation or polyamines. The polyamines, spermine and spermidine, were most effective leading to 3-fold higher rates of nucleotide incorporation than could be obtained at any concentration of the other cations. However, the polyamines were not an absolute requirement for enzyme activity. The polyamine stimulation of tRNA nucleotidyltransferase was not due to alteration of the pH or ionic strength of the reaction mixture or reactivation of denatured tRNA or enzyme. Polyamines also had no effect on the apparent Km values of the substrates. Spermine increased the specificity of the enzyme for tRNA substrates and also inhibited the reverse action. Our results suggest that polyamines may be the normal counterions for tRNA in vivo, and that they affect the rate-limiting step in tRNA nucleotidyltransferase catalysis.