An experimental data handling system has been created as an update to the previous Nucleolar Proteome Database (NOPdb3.0: http://www.lamondlab.com/NOPdb3.0/). This updated system is able to manage large data sets identified by multiple mass spectrometry and has been used to analyse highly purified preparations of human nucleoli from different cell lines. The newly created application includes a dynamic relational database, which is kept up to date by laboratory staff. The data are further annotated with information from specific external sources on the web, including the IPI and Gene Ontology databases. In addition, an Application Programming Interface provides external users with a portal to link into the nucleolar proteome database and hence, gain access to continually updated results. From the initial similar to 700 human proteins identified in the previous iteration of the NOPdb, we have now identified over 50 000 peptides contained in over 4500 human proteins from purified nucleoli, providing enhanced coverage of the nucleolar proteome.