Eukaryotes synthesize queuosine (nucleoside Q) by the irreversible base-for-base exchange of queuine (Q base) for guanine at tRNA position 34, a reaction catalyzed by tRNA-guanine transglycosylase (TGT). The physiological role of Q remains unknown but the tRNA of tumor cells often is undermodified with respect to Q. Toward an understanding of the function of Q in normal and neoplastic cells we have isolated and characterized the cDNA for rabbit TGT. Rabbit erythrocyte TGT was reported previously to be a dimer of 60- and 43-kDa subunits (N. K. Howes and W. R. Farkas, 1978, J. Biol. Chem. 253, 9082-9078). Here we present the cDNA sequence for the apparent 60-kDa subunit; it contains an open reading frame encoding a 493-residue protein. The rabbit TGT 60-kDa subunit shares significant sequence similarity with the deubiquitinating enzyme family (F. R. Papa and M. Hochstrasser, 1993, nature 366, 313-319), especially with sequence elements that include conserved Cys and His residues.