Reproducible conditions have been developed for crystallization of transfer RNA. The conditions may be applicable to many pure transfer RNA species since identical procedures (except for initial transfer RNA concentration) yielded good crystals from both yeast and Escherichia coli transfer RNA. These crystals, which must be kept at temperatures below about 10°C and handled in vapor of controlled alcohol concentration, have been studied by x-ray crystallography. The availability of crystals of a nucleic acid opens a route for extending knowledge of the tertiary structure of transfer RNA and its relation to important biological functions.