A genetic locus is described that specifies two Saccharomyces cerevisiae small nuclear RNAs (snRNAs). The genes specifying the two snRNAs are separated by only 67 base pairs and are transcribed in the same direction. The product RNAs contain 128 and 190 nucleotides and are designated snR128 and snR190, respectively. These RNAs resemble snRNAs of other eucaryotes in nuclear localization and possession of a 5' trimethylguanosine cap. Neither snRNA is related in sequence to previously described vertebrate or yeast snRNAs. Both RNAs exhibit properties consistent with nucleolar organization and hydrogen bonding to pre-rRNA species, suggesting possible roles in ribosome biogenesis. The snR128 species cosediments with deproteinized 27S pre-rRNA, whereas snR190 is associated with a 20S intermediate. Gene disruption in vitro followed by replacement of the chromosomal alleles reveals that SNR128 is essential, whereas SNR190 is not.