The continued widespread use of malathion in Aedes aegypti control programs in Latin America has generated insecticide resistance to this chemical in Culex quinquefasciatus but not in Ae. aegypti. To determine the extent of this resistance, the susceptibility of Cx. quinquefasciatus and Ae. aegypti from several countries to malathion was evaluated. Bioassay results indicated that all Ae. aegypti strains evaluated from Cuba, Venezuela, Costa Rica, and Jamaica were susceptible to malathion in spite of the historical use of this insecticide in Ae. aegypti control programs in these countries. In contrast, a high level of resistance to this insecticide was found in Cx. quinquefasciatus from Venezuela, Colombia, Brazil, and Cuba. Synergist assays indicated that neither esterases nor mixed-function oxidases (MFOs) were involved as the resistance mechanism to malathion in any of the Ae. aegypti strains tested. In Cx. quinquefasciatus, synergist assays confirmed that esterases played an important role in malathion resistance but MFOs were not involved in causing malathion resistance in this species. Biochemical assays showed that both resistance mechanisms were present in the Ae. aegypti and Cx. quinquefasciatus populations. Acrylamide electrophoresis gels revealed that all Ae. aegypti strains had a strongly staining, clear band, named A4, and had a relative mobility (Rm) value of 0.7. Analysis if the results of this study suggested that malathion could continue to be used for the emergency control of Ae. aegypti, the mosquito vector for dengue and dengue hemorrhagic fever in the Americas, but that malathion is probably not effective for the control of adult Cx. quinquefasciatus in urban areas. Therefore, control operations should integrate nonorganophosphate insecticides such as pyrethroids for control of these 2 species found in the urban environment.