The processing of intermediates of Physarum rRNA has been investigated using two cloned rDNA restriction fragments as sequence specific probes. The size of the largest stable pre-rRNA detected is 11.8 Kb, as determined by denaturing gel electrophoresis. R-loop analysis revealed that this pre-rRNA fraction mainly consists of transcripts which do not contain the two insertion sequences present in the 26 S gene. However, a small number of molecules are found which still contain one or the other, or both, of these introns. These observations suggest that the two introns are transcribed but spliced out in a random order before RNA synthesis has reached the 3-end of the primary transcript. Transcription starts at about 17.7 Kb and stops at 4.4 Kb as measured from the ends of the linear palindromic rDNA molecule. Besides the 11.8 Kb transcript several further ribosomal RNA precursors have been identified, ranging in size from 2.5 to 8.8 Kb. In addition, the genes for 5.8 S RNA have been located by R-loop mapping.