Using simian immunodeficiency virus (SIV) infection of rhesus macaques to model human immunodeficiency virus (HIV) infection of humans, we assessed whether broadly reactive vaccine-induced humoral immunity would remain broadly reactive after viral challenge, and whether there would be significant differences in anamnestic antibody responses if animals were challenged when predominately effector or memory lymphocyte populations were present. Animals immunized over a prolonged period and challenged 11 months after vaccination mounted more broadly reactive and stronger humoral immunity than those rapidly vaccinated and challenged 2 weeks after their final vaccinations. These data suggest that vaccination schedule and the timing of virus challenge should be considered when evaluating future candidate HIV vaccines.