The effects of ionizing radiation (137Cs) on processing of ribosomal RNA (rRNA) were studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. The 45 S rRNA precursor, and its two major daughter species, 28 and 18 S rRNA, were separated by gel electrophoresis and the extent of radiolabel incorporation into each was determined at various times after irradiation. This approach permitted kinetic analysis of processing of the 45 S rRNA which had been predominantly synthesized (radiolabeled) prior to irradiation. Since they both derive from the same 45 S pre-rRNA transcript, 28 and 18 S rRNA are produced with a stoichiometry of 1:1, as observed in control cells in the present studies. However, within 1 h following 10 Gy an altered stoichiometry of 28 S:18 S rRNA was apparent, reaching 1.6:1 by 5-7 h following irradiation. This alteration was also observed following the higher dose of 20 Gy, but not following exposures of 5 Gy or less. The 18 S portion of the 45 S pre-rRNA is transcribed prior to the 28 S portion. Consequently, an increase in the 28 S/18 S ratio can only be due to degradation of the 18 S species during or after processing. This alteration may represent a response to radiation-induced growth arrest, by reducing the number of newly synthesized ribosomes that would otherwise be required for cell propagation.