Abstract: There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis.