Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRNA transcription regulation have been elucidated in great detail, the functional organization of the multicopy rRNA gene clusters (rDNA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SIM) to investigate the spatial organization of transcriptionally competent active rDNA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRNA processing. Simultaneous imaging of all active genes bound by Pol I and the architectural chromatin protein Upstream Binding Transcription Factor (UBF) reveals a random spatial orientation of regular repeats of rDNA coding sequences within the nucleoli. These observations imply rDNA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. Collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail.