The advent of next-generation sequencing technologies has greatly impacted genomic research by providing a means for increasing the amount of sequence information in a cost effective fashion. Among the major next-generation sequencing technologies, the Roche 454 platform has been widely adopted in HIV research. We discuss a broad range of applications of the 454 pyrosequencing platform in the HIV field, with a particular emphasis on antiretroviral therapy and virus-host interaction-related research. We also highlight some of the bioinformatics challenges, as well as advantages and potential limitations of this “deep” sequencing tool, and hint at future research applications. (AIDS Rev. 2010;12:89-102) Corresponding author: Bram Vrancken, bram.vrancken@rega.kuleuven.be