Anopheles pseudopunctipennis, one of the primary vectors of malaria in the southwest of Colombia, was evaluated for susceptibility to the 3 major insecticide groups (organophosphates, pyrethroids, and carbamates) by bioassay and biochemical assay. Larval populations, which were collected principally from irrigation channels in agricultural areas, where the intensity of insecticide use varied, were utilized to establish susceptibility for the 1st time in this species. The baselines for each population showed a range of biological susceptibility to the insecticides evaluated, but overall no resistance was detected according to standards established by the World Health Organization. The high sensitivity of biochemical microassays enabled the detection of a small proportion of mosquitoes with higher levels of nonspecific esterases and mixed-function oxidases from 2 areas where agricultural application of organophosphate and pyrethroid insecticides had been heavy. These differences were not sufficient to affect susceptibility as measured by bioassay. No evidence of insensitive acetylcholinesterase was observed. Absence of resistance in areas that have experienced heavy insecticide application could be explained by genetic drift, by gene flow from areas without insecticide pressure, by manner of exposure to the insecticides, or by recent changes in agricultural activities that decreased insecticide use. Baseline values were established that serve as provisional susceptibility thresholds for applying simple Centers for Disease Control and Prevention biochemical assay and bioassay methods to larvae of this anopheline species.