The development and approval of the first CCR5 antagonist, maraviroc, which exclusively acts against CCR5-tropic variants of HIV, has necessitated the determination of HIV coreceptor usage in the clinical setting. Although the phenotypic assay Trofile® (Monogram Biosciences, San Francisco, CA, USA) has been the most widely used diagnostic test, it remains far from perfect for clinical purposes. Genotypic methods represent a more feasible alternative to phenotypic assays. Several rules and algorithms based on V3 gene sequences have been developed to predict HIV coreceptor usage. Even though their sensitivity in detecting CXCR4-using variants was considered to be low in initial studies, more recent data using improved interpretation systems have shown that these rules and algorithms may accurately guide the use of CCR5 antagonists. Consideration of CCR5 antagonist therapy in clinical situations other than salvage therapy (e.g. switch or intensification strategies) has required the examination of coreceptor tropism in proviral DNA or stored plasma samples before patients achieve undetectable viremia with their current therapy. V3 genotyping either in older stored viremic plasma samples or in proviral DNA from aviremic specimens seems to confidently guide CCR5 antagonist therapy. Thus, a much broader use of this unique antiretroviral drug class should be anticipated following a wider use of genotypic tropism determination. J Viral Entry 2010;4(1):12–20.