Natural populations of Anopheles albimanus, An. darlingi and An. nuneztovari were tested for their irritability to DDT in different regions of Colombia. Individual anophelines were exposed to filter papers impregnated with 2% DDT and irritability was determined based on the number of flights a mosquito made in 15 min. All three species showed variability in their irritability to DDT. Anopheles albimanus showed irritability only in Bolivar, but not in three other regions. Anopheles darlingi showed no irritability in neither the Atlantico nor Llanos regions, while An. nuneztovari showed irritability in the Oriente but not in Uraba. The irritability to DDT shown in some populations of An. albimanus and An. nuneztovari may reduce the effectiveness of residual applications of this insecticide by causing the mosquitoes to seek untreated surfaces and/or leave the house to rest outdoors and thus avoid a lethal dose.