Abstract Motivation: The large and growing body of experimental data on biomolecular binding is of enormous value in developing a deeper understanding of molecular biology, in developing new therapeutics, and in various molecular design applications. However, most of these data are found only in the published literature and are therefore difficult to access and use. No existing public database has focused on measured binding affinities and has provided query capabilities that include chemical structure and sequence homology searches. Methods & results: We have created Binding DataBase (BindingDB), a public, web-accessible database of measured binding affinities. BindingDB is based upon a relational data specification for describing binding measurements via Isothermal Titration Calorimetry (ITC) and enzyme inhibition. A corresponding XML Document Type Definition (DTD) is used to create and parse intermediate files during the on-line deposition process and will also be used for data interchange, including collection of data from other sources. The on-line query interface, which is constructed with Java Servlet technology, supports standard SQL queries as well as searches for molecules by chemical structure and sequence homology. The on-line deposition interface uses Java Server Pages and JavaBean objects to generate dynamic HTML and to store intermediate results. The resulting data resource provides a range of functionality with brisk response-times, and lends itself well to continued development and enhancement. Availability: The BindingDB is publicly available at http://www.bindingdb.org. The database schema and DTD file are available at http://www.bindingdb.org/bind/entity˙report.html, and http://www.bindingdb.org/bind/deposition/xml/BindingDB.xml, respectively. Contact: [email protected] * To whom correspondence should be addressed. 2 Present address: NovaScreen, 7170 Standard Drive, Hanover, MD 21076, USA.