We have developed PathBuilder, an open-source web application to annotate biological information pertaining to signaling pathways and to create web-based pathway resources. PathBuilder enables annotation of molecular events including protein-protein interactions, enzyme-substrate relationships and protein translocation events either manually or through automated importing of data from other databases. Salient features of PathBuilder include automatic validation of data formats, built-in modules for visualization of pathways, automated import of data from other pathway resources, export of data in several standard data exchange formats and an application programming interface for retrieving existing pathway datasets.