The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1). ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites [( rho-]) at high frequency, and showed signs of aberrant cell division. A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs.