We have characterized a new delayed-early response mRNA encoding a 21-kDa product (MRPL12) that accumulates during the G1 phase of growth-stimulated cells. MRPL12 is the mammalian homologue to chloroplastic and bacterial L12 ribosomal proteins. Immunofluorescence microscopy and cell fractionation indicate a predominant mitochondrial localization in various mammalian cell lines. The NH2-terminal 49 amino acids are necessary and sufficient to target the protein within the mitochondria and are probably cleaved off during import. MRPL12 proteins associated in vitro and cofractionate with ribosomal structures, as is the case for prokaryotic L12 proteins. Expression of a dominant inhibitory truncated protein leads to a severe reduction in cell growth by inhibiting mitochondrial ATP production. MRPL12 is the first mammalian mitochondrial ribosomal protein to be characterized.