Proteinortho is a software tool designed for large-scale orthology analysis, particularly useful for comparing hundreds of species with millions of proteins. It addresses the challenges of memory consumption and computational efficiency in traditional orthology detection methods. The tool implements an extended version of the reciprocal best alignment heuristic, which is adapted to handle distributed computing on multi-core hardware. Proteinortho reduces memory requirements significantly compared to existing tools, making it feasible to perform genome-wide orthology analyses on standard hardware. The authors applied Proteinortho to the complete set of 717 eubacterial genomes available at NCBI in 2009, identifying 30 proteins present in 99% of all bacterial proteomes. The tool's performance and memory efficiency were benchmarked against OrthoMCL, demonstrating superior speed and reduced memory usage. Proteinortho's effectiveness is further validated through its ability to identify domain-wide conserved proteins, which can be useful for phylogenetic studies and species identification.Proteinortho is a software tool designed for large-scale orthology analysis, particularly useful for comparing hundreds of species with millions of proteins. It addresses the challenges of memory consumption and computational efficiency in traditional orthology detection methods. The tool implements an extended version of the reciprocal best alignment heuristic, which is adapted to handle distributed computing on multi-core hardware. Proteinortho reduces memory requirements significantly compared to existing tools, making it feasible to perform genome-wide orthology analyses on standard hardware. The authors applied Proteinortho to the complete set of 717 eubacterial genomes available at NCBI in 2009, identifying 30 proteins present in 99% of all bacterial proteomes. The tool's performance and memory efficiency were benchmarked against OrthoMCL, demonstrating superior speed and reduced memory usage. Proteinortho's effectiveness is further validated through its ability to identify domain-wide conserved proteins, which can be useful for phylogenetic studies and species identification.