The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1-to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.Human immunodeficiency virus type 1 (HIV-1) infection is usually initiated by outgrowth of one or a few genotypic variants (42,44,59,72,75,77) followed by a prolonged period of asymptomatic infection during which virus populations diversify and after a period (26) diverge from the founder sequence (61). This process is driven by the high rate of virus genetic mutation and recombination (46, 62) and the selective forces on the diverse virus population imposed by the host (48). Evolution of HIV-1 envelope gene (env) sequences leads to expansion of coreceptor use/cell tropism from CCR5 (macrophage tropism) to CXCR4 (T-cell line tropism) in Ն50% of subtype B-infected individuals at late stages of disease (3,47,60,61,65,71). Infection with subtype C HIV-1 is associated with a lower incidence of coreceptor switching (8,54,56), and infection with subtype D may be associated with more-frequent switching (29). This switch in coreceptor use correlates with more-rapid clinical progression of disease (2, 12, 35), although this correlation does not establish causation.In vitro studies on coreceptor switching, as well as genetic analysis of viral env sequences, suggest that viruses at intermediate stages between CCR5 use and CXCR4 use (the transition from R5 to R5X4 or X4 virus) are less fit than parental R5 populations (49,50,52,31,73). Intermediate stages in coreceptor switching may also differ in preference for CCR5 versus CXCR4, and these stages have recently been proposed to be designated dual-R (CCR5 preference) or dual-X (CXCR4 preference) based on cell line entry assays (29). These presumably less fit intermediates are often d...