The eucaryotic tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) exchanges a guanine residue in the anticodon of tRNAs specific for aspartic acid, asparagine, histidine and tyrosine with the nutritionally derived deazaguanine base queuine (q), and with queuine precursors and guanine. In higher eucaryotes, the amount of the resulting queuosine nucleoside (Q) is dependent on the developmental state of the respective cells. Neoplastically transformed and fast-proliferating cells usually are almost Q-deficient. The Tgt enzyme from bovine liver was purified 14,000-fold by DEAE cellulose chromatography, ammonium sulfate precipitation, and two subsequent affinity chromatography steps on heparin and tRNA agarose. The purest preparations contained two major proteins of 66 kDa and 32 kDa as revealed by SDS/PAGE and silver staining. The Km of the Tgt enzyme for guanine was 1.4 microM and the value for a purified Q-specific tRNA(Tyr), was 0.08 microM. The enzyme was active over a broad pH range; the activity was independent of metal ions and was strongly inhibited by salt concentrations higher than 50 mM. The determination and comparison of the N-terminal amino acid sequences from endoproteinase Lys-C cleavage products of the two subunits revealed no significant similarity to any known proteins.