We have identified an RNA species that appears to be induced by oxidative stress in hamster HA-1 fibroblasts using the differential display technique, but instead is found to be degraded when evaluated by Northern blot hybridization. Cloning and subsequent sequencing identified the partially degraded RNA as 16S ribosomal RNA (rRNA), a major component of mitochondrial ribosomes. Degradation, and associated decreases in the levels of the mature- and precursor-species of 16S rRNA, appear to be dependent upon calcium, but not cytoplasmic protein synthesis nor nuclear transcription. Other decreased mitochondrial RNAs were also identified, including 12S rRNA, NADH dehydrogenase subunit 6, ATPase subunit 6, and cytochrome oxidase subunits I and III. A significant part of many, if not all, of these RNA decreases was due to degradation. As compared with 16S rRNA, significantly less degradation was observed for cytoplasmic 28S/18S rRNAs, even at very high peroxide concentration. Analysis of 21 cytoplasmic mRNAs revealed little or no decrease in mature band signal in response to peroxide, and several cytoplasmic mRNAs were actually up-regulated. Thus, a preferential down-regulation of mitochondrial RNAs occurs in HA-1 fibroblasts in response to hydrogen peroxide. Subcellular fractionation analysis, using 16S rRNA degradation as a gauge, indicates that this down-regulation is specific to mitochondria. The down-regulation of mitochondrial RNAs may represent a general mechanism by which cells protect themselves against oxidative stress.