The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.