Cell-free extracts prepared from S. cerevisiae cells were incubated in the presence of [α-32P]-labeled ATP, CTP, GTP or UTP. An RNA larger than ribosomal 25S RNA with an apparent size of approximately 30S was prominently labeled on its 3′ end in the presence of ATP or UTP but not with CTP or GTP. This labeled RNA was not hybrid-selected by cloned yeast ribosomal DNA; in addition, this ∼30S RNA was not cleaved by RNase H in the presence of complementary deoxyribooligonucleotides to rRNA. These two lines of evidence show that this ∼30S RNA is not structurally related to ribosomal RNA gene repeat. The cell-free extracts prepared from yeast cells containing temperature-sensitive poly(A) polymerase adenylated this novel yeast RNA at restrictive temperature with efficiency similar to extracts prepared from wild-type yeast cells. These data show that the enzyme responsible for adenylation of this ∼30S RNA is distinct from mRNA poly(A) polymerase. While the human SRP RNA 3′ adenylating enzyme in the HeLa cell extract adenylated human SRP or Alu RNAs, the yeast adenylating enzyme did not adenylate the human SRP or Alu RNAs in vitro; these data indicate species specificity for this adenylating enzyme.