Mosquitoes were collected in the Amazon Basin, near Iquitos, Peru, and used in experimental studies to evaluate their susceptibility to strains of eastern equine encephalitis virus (EEEV) that were isolated from mosquitoes captured within 20 km of Iquitos. When fed on hamsters or chickens with a viremia of ≈10⁵ plaque-forming units (PFU) of EEEV/ml, Culex pedroi Sirivanakarn and Belkin, Aedes fulvus (Wiedemann), Psorophora albigenu (Peryassu), and Psorophora ferox (Von Humboldt) were susceptible to infection, whereas none of the Aedes serratus (Theobald), Culex vomerifer Komp, Culex gnomatos Sallum, Huchings, and Ferreira, Culex portesi Senevet and Abonnenc, or Culex coronator Dyar and Knab became infected, even though they fed on the same viremic blood sources. When these mosquito species fed on animals with viremias of ≈10⁸ PFU/ml, Cx. pedroi, Ae. fulvus, Ps. albigenu, and Psorophora cingulata (Fabricius) were the most susceptible. Mosquito species were susceptible to both a lineage II (Brazil-Peru) and a lineage III (Argentina-Panama) isolate of EEEV. This study, combined with the repeated isolation of strains of EEEV from Cx. pedroi captured in the Amazon Basin region of Peru, suggests that Cx. pedroi may be the primary enzootic vector of EEEV in this region.