MitoCarta2.0 is an updated inventory of mitochondrial proteins in humans and mice, providing a comprehensive list of genes encoding proteins with strong evidence of mitochondrial localization. The database was constructed using a combination of experimental and computational approaches, including mass spectrometry, literature curation, homology mapping, and Bayesian integration. The updated version includes 1158 human genes and 1158 mouse genes, with significant improvements in gene models, proteomic data, and homology detection methods. The MitoCarta2.0 database is freely available online and provides detailed information on protein expression across 14 mouse tissues, making it a valuable resource for studying mitochondrial function and disease.MitoCarta2.0 is an updated inventory of mitochondrial proteins in humans and mice, providing a comprehensive list of genes encoding proteins with strong evidence of mitochondrial localization. The database was constructed using a combination of experimental and computational approaches, including mass spectrometry, literature curation, homology mapping, and Bayesian integration. The updated version includes 1158 human genes and 1158 mouse genes, with significant improvements in gene models, proteomic data, and homology detection methods. The MitoCarta2.0 database is freely available online and provides detailed information on protein expression across 14 mouse tissues, making it a valuable resource for studying mitochondrial function and disease.