We have determined the relationship between overall nuclear architecture, chromosome territories, and transcription sites within the nucleus, using three-dimensional confocal microscopy of well preserved tissue sections of wheat roots. Chromosome territories were visualized by GISH using rye genomic probe in wheat/rye translocation and addition lines. The chromosomes appeared as elongated regions and showed a clear centromere-telomere polarization, with the two visualized chromosomes lying approximately parallel to one another across the nucleus. Labeling with probes to telomeres and centromeres confirmed a striking Rabl configuration in all cells, with a clear clustering of the centromeres, and cell files often maintained a common polarity through several division cycles. Transcription sites were detected by BrUTP incorporation in unfixed tissue sections and revealed a pattern of numerous foci uniformly distributed throughout the nucleoplasm, as well as more intensely labeled foci in the nucleoli. It has been suggested that the gene-rich regions in wheat chromosomes are clustered towards the telomeres. However, we found no indication of a difference in concentration of transcription sites between telomere and centromere poles of the nucleus. Neither could we detect any evidence that the transcription sites were preferentially localized with respect to the chromosome territorial boundaries.