The localization of transcribing rRNA genes within nucleoli of mammalian cells, although intensively studied, has not been established. Most published papers on this topic situate transcribing ribosomal genes either to nucleolar fibrillar centers or to nucleolar dense fibrillar components. To clarify this point, we have generated the electron microscopic affinity cytochemistry picture of the nucleolus of cultured mammalian cells. Three kinds of affinity probes have been used: (1) probes to nucleolar chromatin, including rDNA sequences; (2) probes to a number of macromolecules (such as RNA polymerase I) which are directly, or indirectly, involved in the synthesis and processing of rRNA and formation of preribosomes; (3) antibodies to bromouridine for a recently standardized nonisotopical method depicting incorporated bromouridine within RNA. The results suggest the localization of transcription sites not only to dense fibrillar components but also to the border region between these components and fibrillar centers. Our data support a hypothesis that in metabolically active mammalian nucleoli, fibrillar centers and dense fibrillar components form a single functional domain for the transcription of rRNA genes, with nascent transcripts generating ''automatically'' dense fibrillar components. Through the active process of transcription, individual rRNA genes thus become engulfed within dense fibrillar components. (C) 1995 Academic Press, Inc.