The success of gene therapy depends in part on our understanding of the biology of gene therapy vectors. This knowledge must be used to improve the function, safety, and versatility of the vector system. For decades, we have known which viral proteins are involved in formation of the adenovirus (Ad) capsid, but we are still learning how these proteins can be altered or manipulated to improve vector function. The Ad protein IX (pIX) was originally identified as a minor component of the Ad capsid, but was not essential for virion formation. However, more recent studies have suggested that pIX may have multiple roles in the Ad life cycle, including acting as a transcriptional activator and reorganizing nuclear proteins to provide an environment more conducive to virus replication. In gene therapy studies, removal of pIX from the Ad vector backbone was used to increase the cloning capacity of E1-deleted Ad vectors and to develop a new method for preparing helper-dependent Ad vectors. pIX has also been at the center of numerous attempts to eliminate the problem of replication-competent Ad in Ad vector preparations. Finally, pIX represents a versatile platform for the presentation of polypeptides on the surface of the viral capsid, including ligands for virus retargeting and fluorescent proteins for visualizing the virus in vitro and in vivo. Thus, the importance and uses of this "minor" capsid protein have changed significantly over the past few years.