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.