Pathway Tools is a bioinformatics software environment with a broad set of capabilities. The software provides genome-informatics tools such as a genome browser, sequence alignments, a genome-variant analyzer and comparative-genomics operations. It offers metabolic-informatics tools, such as metabolic reconstruction, quantitative metabolic modeling, prediction of reaction atom mappings and metabolic route search. Pathway Tools also provides regulatory-informatics tools, such as the ability to represent and visualize a wide range of regulatory interactions. This article outlines the advances in Pathway Tools in the past 5 years. Major additions include components for metabolic modeling, metabolic route search, computation of atom mappings and estimation of compound Gibbs free energies of formation, addition of editors for signaling pathways, for genome sequences and for cellular architecture, storage of gene essentiality data and phenotype data, display of multiple alignments, and of signaling and electron-transport pathways, and development of Python and web-services application programming interfaces. Scientists around the world have created more than 9800 Pathway/Genome Databases by using Pathway Tools, many of which are curated databases for important model organisms.