Compared to other tRNAs all known histidine tRNAs have the unique feature of possessing an additional nucleotide at their 5' end. It is usually a guanosine residue but not in bacteriophage T5 tRNA which carries an additional uridine. The additional nucleotide is not encoded in eukaryotic histidine tRNA genes but is added in a post-transcriptional modification reaction (Cooley, L., Appel, B., and Söll, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6475-6479) by histidine tRNA guanylyltransferase (tRNAHis guanylyltransferase). Here we report the purification of this enzyme from Saccharomyces cerevisiae and the determination of some of its physical properties. Six different steps including Polymin P precipitation, chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, ATP-agarose, and gel filtration on Superose 12 were employed for the purification of the guanylyltransferase from an S-100 extract. A Stokes radius of 46.5 +/- 0.5 A and a sedimentation coefficient (S20,w) of 7.8 +/- 0.2 were determined by gel filtration and rate zonal sedimentation, respectively. A relative molecular weight (Mr) of approximately 120,000 was calculated for the purified native enzyme. The Mr of the denatured protein is approximately 58,000 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate a homodimeric (alpha 2) structure for the enzyme. Among all acceptor RNAs in unfractionated tRNA only tRNAHis is a substrate for the purified guanylyltransferase. The reaction requires ATP, a guanosine substrate, and a divalent metal ion. Treatment of guanylyltransferase with 5,5'-dithiobis(2-nitrobenzoic)-acid abolishes activity; this suggests the importance of sulfhydryl groups for enzymatic activity. The enzyme shows discrimination among different histidine tRNA species; tRNA from plant and prokaryotes are better substrates than mammalian and insect tRNAs.