A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53؉/؉ and p53؊/؊ mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53؊/؊ mice. mc3s5/mtCLIC mRNA and protein are higher in p53؉/؉ compared with p53؊/؊ basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor ␣ also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor ␣.The multiple functions now ascribed to the p53 tumor suppressor gene include cell cycle control, DNA repair, senescence, differentiation, and apoptosis (reviewed in Refs. 1-3). p53 exerts part of its biological function by activating or repressing the transcription of several target genes (2, 4, 5). p53 is particularly important in the skin because it is frequently mutated in preneoplastic and neoplastic human skin lesions (6), and loss of p53 enhances malignant progression of experimentally induced murine skin tumors (7). In keratinocytes, p53-dependent transcriptional activity increases in association with terminal differentiation (8). However, the skin of p53-deficient mice appears to be normal in vivo (9), and the expression of differentiation markers by p53Ϫ/Ϫ keratinocytes in vitro cannot be distinguished from that of wild-type (8). This implies that p53 is dispensable for normal skin development and differentiation. However, deletion of one or both p53 alleles enhances the establishment of immortal keratinocyte cell lines in vitro (10), suggesting that p53 could participate in keratinocyte mortality in a more subtle way, not involving expression of the major differentiation-related markers. To detect such a con...