Simian immunodeficiency virus (SIV) has been shown to progress through a number of changes that lead to the emergence of pathogenic viral variants in macaques initially infected with a mildly cytopathic variant, SIVMneCL8. One of these late-stage isolates, SIVMne170, replicates to high levels in vivo and causes a rapid disease course when reintroduced into naïve macaques, resulting in a viral set point up to 3,000-fold higher than the set point of the parental virus, SIVMneCL8. However, in cell culture both viruses replicate with similar kinetics. One major difference between in vivo and in vitro cultures is the life span of the infected cells. Here, we manipulated the life span of infected cells in vitro, and we show that the fitness of SIVMne170 in cultures with a limited cell life span dramatically increased compared to its fitness in cultures with a nonlimited life span of cells. The increase in fitness was at least partially due to the fact that the rapid turnover system eliminates the negative influence of the cytopathic effects associated with replication of SIVMne170. Because the relative fitness of SIVMneCL8 and SIVMne170 observed in the rapid turnover system more accurately reflects their fitness in vivo, the system represents an improved approach to comparing relative fitness of viruses.