The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.