Mitochondria play pivotal roles in the metabolism and physiology of eukaryotic cells. The composition of mitochondria varies in different cell types, and therefore it is crucial to know the set of proteins that constitutes the organelle in each cell type. The identification of mitochondrial proteins has been furthered by biochemical methods, but half of mitochondrial proteins still remain unidentified. We have developed a genetic method to identify mitochondrial proteins with reconstitution of split enhanced green fluorescent protein (EGFP) by protein splicing. cDNA are randomly fused to the N-terminal half of EGFP and are introduced into cells expressing the C-terminal EGFP in the mitochondrial matrix. If the cDNA encodes a protein that is targeted into mitochondria, full-length EGFP is reconstituted in the mitochondrial matrix by protein splicing. The fluorescent cells are collected by fluorescence-activated cell sorting and the cDNA are identified by DNA sequencing. This method provides a means to map proteins distributed within mitochondria of different mammalian cells.