Wheat embryo ribosomal RNA (rRNA) has been shown by optical rotatory dispersion (o.r.d.) and absorption–temperature measurements to possess a substantial secondary structure in solution which is stabilized by hydrogen bonding of paired bases in helical segments, and by nearest-neighbor base stacking in single-stranded segments of the molecule. Experimental conditions were established where single-strand stacked conformations predominated over base-paired regions. This state of affairs was effected by reducing the number of hydrogen-bonded nucleotides in RNA by any of three treatments: protonation, formylation, or reduction of the ionic strength of the medium to essentially zero. At the same time, use of the organic solvent ethylene glycol, which preferentially breaks hydrophobic bonds, has demonstrated that base stacking plays a significant role, as well, in the stabilization of the native conformation. Application of the empirical method of C. R. Cantor, S. R. Jaskunis, and I. Tinoco (J, Mol. Biol. 20, 39 (1966)), whereby it is possible to predict the optical rotatory properties of single-stranded polynucleotides from the corresponding properties of oligonucleotides, successfully demonstrated that wheat embryo rRNA, in the absence of salt, is predominantly in a single-stranded stacked conformation. From hyperchromicity data measured on RNA under conditions where hydrogen-bonding and base-stacking forces were delineated, the helical content of wheat embryo rRNA has been estimated as 65%. In view of the close similarity in the o.r.d. curves of RNA in salt solution and in the ribosome, it is concluded that RNA in the ribosome must be in a largely base-paired double-helical conformation, joined by single-stranded links.