We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5' region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli, (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5' end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3' end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions - fibrillar centers, dense fibrillar components, and granular components - for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.