Background. Over recent years, treatment guidelines for human immunodeficiency virus (HIV) infection have evolved from monotherapy to combination regimens that include ≥3 active drugs, resulting in a sharp decrease in morbidity and mortality. In the present article, we evaluated changes in HIV type 1 viral fitness associated with the sequential introduction of antiretroviral treatment strategies in 4 chronically infected patients with sustained CD4 cell count despite having a persistently detectable viral load. Methods. Plasma samples were obtained before and during treatment to construct recombinant virus containing the 3′-end of gag, the protease and the reverse-transcriptase coding region. Drug susceptibility phenotype was evaluated with a panel of multiple reverse-transcriptase and protease inhibitors. Replicative capacity (RC) and infectivity were measured, and production of p24 was monitored after transfection. Results. Multidrug-resistant (MDR) viruses selected during long-term antiretroviral therapy were less fit and infectious than their wild-type or monotherapy-selected counterparts, with the exception of viruses recovered from patient B. In 3 of 4 cases, p24 kinetics after transfection showed a delay in viral production of recombinant viruses containing MDR mutations. Data from the RC and infectivity assays showed good correlation (P < .03) and corroborated the p24 kinetics data. Conclusions. This study shows that accumulation of MDR mutations during long-term antiretroviral treatment results, albeit not in all cases, in reductions of viral fitness. © 2005 by the Infectious Diseases Society of America. All rights reserved.