The aim was to develop quantitative estimates of farmers' pesticide exposure to atrazine and to provide an overview of background levels of selected non-persistent pesticides among corn farmers in a longitudinal molecular epidemiologic study. The study population consisted of 30 Agricultural Health Study farmers from Iowa and 10 non-farming controls. Farmers completed daily and weekly diaries from March to November in 2002 and 2003 on pesticide use and other exposure determinants. Urine samples were collected at 10 time points relative to atrazine application and other farming activities. Pesticide exposure was assessed using urinary metabolites and diaries. The analytical limit of detection (LOD) ranged between 0.1 and 0.2 microg/l for all pesticide analytes except for isazaphos (1.5 microg/l) and diazinon (0.7 microg/l). Farmers had higher geometric mean urinary atrazine mercapturate (AZM) values than controls during planting (1.1 vs <LOD microg/g creatinine; P<0.05). AZM levels among farmers were significantly related to the amount of atrazine applied (P=0.015). Interestingly, farmers had a larger proportion of samples above the LOD than controls even after exclusion of observations with an atrazine application within 7 days before urine collection (38% vs 6%, P<0.0001). A similar pattern was observed for 2,4-D and acetochlor (92% vs 47%, P<0.0001 and 45% vs 4%, P<0.0001, respectively). Urinary AZM levels in farmers were largely driven by recent application of atrazine. Therefore, the amount of atrazine applied is likely to provide valid surrogates of atrazine exposure in epidemiologic studies. Elevated background levels of non-persistent pesticides, especially 2,4-D, indicate importance in epidemiologic studies of capturing pesticide exposures that might not be directly related to the actual application.