Background: Biological pathways are a useful abstraction of biological concepts, and software tools to deal with pathway diagrams can help biological research. PathVisio is a new visualization tool for biological pathways that mimics the popular GenMAPP tool with a completely new Java implementation that allows better integration with other open source projects. The GenMAPP MAPP file format is replaced by GPML, a new XML file format that provides seamless exchange of graphical pathway information among multiple programs. Results: PathVisio can be combined with other bioinformatics tools to open up three possible uses: Visual compilation of biological knowledge, interpretation of high-throughput expression datasets, and computational augmentation of pathways with interaction information. PathVisio is open source software and available at http://www.pathvisio.org. Conclusion: PathVisio is a graphical editor for biological pathways, with flexibility and ease of use as primary goals. © 2008 van Iersel et al; licensee BioMed Central Ltd.