Because of their history of widespread use in the United States and unknown long-term health effects, organophosphate pesticides (OPs) are being considered as a chemical class of interest in planning for the National Children's Study, a longitudinal study of children's environmental health. The availability and appropriate use of biomarkers to determine absorbed doses of environmental chemicals such as OPs are critical issues. Biomarkers of OP exposure are typically measured in blood and urine; however, postpartum meconium has been shown to be a promising matrix for assessing cumulative in utero exposure to the fetus, and studies are currently in progress to determine the utility of using saliva and amniotic fluid as matrices. In this article, we discuss the advantages and disadvantages of the currently available OP exposure monitoring methods (cholinesterase inhibition in blood, pesticides in blood, metabolites in urine and alternative matrices); study design issues for a large, long-term study of children's environmental health; and current research and future research needs. Because OPs are rapidly metabolized and excreted, the utility of one-time spot measurements of OP biomarkers is questionable unless background exposure levels are relatively stable over time or a specific time frame of interest for the study is identified and samples are collected accordingly. Biomarkers of OP exposure can be a valuable tool in epidemiology of children's environmental health, as long as they are applied and interpreted appropriately.