By combining cryofixation and cryosubstitution in a structural and functional analysis of the nucleus of Saccharomyces cerevisiae, we identified morphological subcompartments in the nucleolus. These were similar to those of nucleoli of higher eukaryotes, such as the fibrillar centre (FC), the dense fibrillar component (DFC) and the granular component (GC). In situ hybridization and immunocytochemistry revealed RNA polymerase I and proteins involved in early steps of ribosomal maturation along the DFC, while the ribosomal genes were detected at the FCs. Our results also suggest that ribosomal transcripts are distributed along a nucleolar network that might include both DFC and GC. We also show that pre-ribosomal subunits may be exported along tracks to the cytoplasm. Export takes place through all the pores of the nuclear envelope, not just those in contact with the nucleolus. Moreover, comparison of the nucleolar organization in S. cerevisiae and in Schizosaccharomyces pombe demonstrated than the distribution of the 5S genes with respect to the 35S transcription unit does not modify the organization of the nucleolus. We also report, for the first time, the ultrastructural localization of RNA polymerase II in yeast. The distribution of RNA polymerase II and morphological details that could be observed in the extra-nucleolar region of cryofixed cells provided cytological evidence of a peripheral region extending along the nuclear envelope that could correspond to heterochromatin in higher eukaryotes.