The specificity of rabbit liver tRNA nucleotidyltransferase with respect to its interaction with acceptor residues at the 3' end of tRNA was analyzed using a model acceptor system consisting of dinucleoside monophosphates or nucleosides. Of all the dinucleoside monophosphates tested, only CpC was an active AMP acceptor, indicating that the specificity of the enzyme conforms exactly to the structure present at the 3' terminus of the natural acceptor, tRNA-C-C. Similarly, CMP incorporation into model acceptors closely paralleled the specificity seen with tRNA-C and tRNA-X. Competition studies between the model acceptors and tRNAs with modified 3' termini suggested that the model compounds bind to the enzyme at the site normally recognizing the 3' terminus of tRNA. Comparison of nucleotide incorporation into tRNAs and into the model acceptors revealed a number of differences which allowed us to separate effects on tRNA structure from direct effects on the reaction. These studies enabled us to distinguish several subsites on the enzyme: an ATP-donor site, two sites specifically recognizing the 2 terminal C residues on tRNA, and a site recognizing the nonreacting part of the tRNA. Thus, these results support several features of the multisite model previously proposed (Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468) to explain tRNA nucleotidyltransferase action.