Organophosphates are pesticides ubiquitous in the environment and have been hypothesized as one of the risk factors for acute lymphoblastic leukemia (ALL). In this study, we evaluated the associations of pesticide exposure in a residential environment with the risk for pediatric ALL. This is a case-control study of children newly diagnosed with ALL, and their mothers (n = 41 child-mother pairs) recruited from Georgetown University Medical Center and Children's National Medical Center in Washington, DC, between January 2005 and January 2008. Cases and controls were matched for age, sex, and county of residence. Environmental exposures were determined by questionnaire and by urinalysis of pesticide metabolites using isotope dilution gas chromatography-high-resolution mass spectrometry. We found that more case mothers (33%) than controls (14%) reported using insecticides in the home (P < 0.02). Other environmental exposures to toxic substances were not significantly associated with the risk of ALL. Pesticide levels were higher in cases than in controls (P < 0.05). Statistically significant differences were found between children with ALL and controls for the organophosphate metabolites diethylthiophosphate (P < 0.03) and diethyldithiophosphate (P < 0.05). The association of ALL risk with pesticide exposure merits further studies to confirm the association.