The purpose of the present study was to investigate if the biological and antigenic properties of human immunodeficiency virus type 2 change over time in cynomolgus macaques (Macaca fascicularis) experimentally infected with HIV-2SBL6669. Sequential virus isolates and serum samples were obtained during a 2-year period and studied in autologous neutralization assays. All six macaques studied seroconverted shortly after infection and remained healthy during the observation period. Virus could be isolated from all six animals during the first 100 days postinfection. Thereafter four monkeys became virus isolation negative, either permanently or transiently (two macaques each), whereas two macaques remained virus isolation positive during the entire observation period. Sequential reisolates from the macaques invariably replicated in HUT-78, U937-2, and Jurkat-tat cell lines, similarly to the HIV-2SBL6669 inoculum virus. The ability to produce neutralizing antibodies correlated with positive virus isolations, hence four macaques produced neutralizing antibodies against inoculum virus and sequential reisolates. Once the neutralizing antibody appeared, sequential reisolates obtained at both early and late time after infection were neutralized, indicating that the neutralizing epitopes of the virus are conserved in the infected animals over time. This is different from the pathogenic SIVsm infection in macaques or HIV-1 infection in humans, where emergence of neutralization resistant variants seems to be the rule. In contrast, in HIV-2-infected macaques the biological properties of the virus are stable and the neutralizing antibody response shows extensive cross-reactivity.