During sporulation of Saccharomyces cerevisiae, most strains accumulate an unmethylated 20S RNA. Contrary to previous reports, this sporulation 20S RNA is distinct from the short-lived methylated 20S RNA precursor of 18S rRNA. This RNA species was found in a cytoplasmic 32S ribonucleoprotein particle consisting of one single-stranded 20S RNA molecule and 18 to 20 identical protein subunits of molecular weight 23,000. The ribonucleoprotein particle was resistant to ribonuclease digestion, although purified 20S RNA was ribonuclease sensitive. Both the RNA and the protein of the 32S ribonucleoprotein particle were only synthesized under conditions that induce sporulation. The accumulation of 20S RNA depended on continued protein synthesis but was actinomycin D insensitive, despite a high guanine-plus-cytosine content. Synthesis of 20S RNA stopped when cells were removed from sporulation conditions and placed in growth medium.