Abstract: Maize (Zea mays L.) has a single pair of 45S rDNA sites that forms secondary constrictions in mitotic cells, and thus presents a simple model for studying the organization and expression pattern of rRNA genes in plants. We analyzed the organization and expression pattern of rRNA genes in root-tip meristematic cells of maize by fluorescence in situ hybridization (FISH), combined PI and DAPI (CPD) staining and silver staining. An 45S rDNA probe revealed two types of signals in all interphase nuclei: strongly fluorescing perinucleolar knobs and less-strongly fluorescing intranucleolar spots. The spots associated with or emanating from knobs were observed in a portion of nuclei. The more spots shown, the smaller are the knobs, and the number of spots was correlated with the activity of interphase cells. Thus, knobs represent condensed inactive rDNA chromatin, and the formation of spots decondensed from knobs is the cytogenetic manifestation of active rRNA gene transcription. The variation in number of spots among nuclei of different stages reflects the difference in number of activated rRNA genes. Sequential CPD and silver staining in interphase and prophase cells revealed that most of the rDNA chromatin did not participate in the formation of the nucleolus. The interphase rDNA FISH results also showed that the level of expression differed between the homologous 45S rDNA sites, which was further confirmed by the difference in length of secondary constriction of homologous chromosomes and differences in the size of homologous Ag-NORs and nucleoli. FISH results showed that the rDNA chromatin in prometaphase cells was relatively decondensed, and silver staining showed the existence of prominent nucleoli in all prometaphase cells and a portion of metaphase cells. These findings indicate that rDNA transcription is active during prometaphase and does not stop until late metaphase.