Abstract To generate a visible reporter of mitochondrial gene expression, we have synthesized a DNA fragment that specifies an enhanced variant of the green fluorescent protein (GFP) in the Saccharomyces cerevisiae mitochondrial genetic code. This reporter gene, GFP m - 3, was inserted into mtDNA at the eighth codon of the COX3 gene. Mitochondria containing this mtDNA could be detected by fluorescence microscopy. Mitochondrially encoded GFP accumulated as soluble matrix protein, whose level could be measured both immunologically and fluorometrically. Quantitation of relative fluorescence by flow cytometry confirmed that cox3∷ GFP m - 3 expression was affected by carbon source and dependent upon COX3 mRNA-specific translational activation. GFP m - 3 will be a valuable tool for studying mitochondrial gene regulation and the intracellular fates of mitochondrially synthesized proteins.