In June 1992 we collected 53 blood plasma samples from nestling red‐winged blackbirds (Agelaius phoeniceus), house sparrows (Passer domesticus), and brown‐headed cowbirds (Molothrus ater) at five study sites in northwestern Minnesota to evaluate the feasibility of using plasma and brain cholinesterase (ChE) activity and reactivation as a means of assessing exposure of nontarget organisms to the operational use of ChE‐ inhibiting insecticides in an agricultural setting. Three sites were adjacent to sugar beet fields that were likely to be treated with chlorpyrifos as Lorsban® (an organophosphorus [OP] insecticide) for control of sugar beet root maggot (Tetanops myopaeformis), and two sites were distant from fields likely to be treated (reference sites). Application of chlorpyrifos in fields surrounding study plots was monitored through contact with landowners and direct observations. Cholinesterase activity levels (total cholinesterase [ChE], acetylcholinesterase [AChE], and butyrylcholinesterase [BChE]) in nestling plasma were measured and tested for reactivation (ChE and AChE) in the presence of 2–PAM, an indication of exposure to an organophosphorus insecticide. In addition, 11 nestlings were euthanatized and in these samples we measured brain ChE activity and reactivation, and we analyzed gastrointestinal tracts and carcass washes for chlorpyrifos residues. Total ChE and BChE activity were lowest in similar‐aged nestlings at sites adjacent to treated beet fields (ChE, t = −2.51, d.f. = 21, p = 0.033; BChE, t = −2.56, d.f. = 21, p = 0.043). Nestlings from sites that were near fields where chlorpyrifos was applied were more likely to exhibit plasma AChE reactivation than nestlings from reference sites where OP or carbamate insecticide application was improbable (x² = 3.805, d.f. = 1, p ∼ 0.05). The magnitude of plasma ChE reactivation was highest within 1 to 3 d of insecticide application, although significant reactivation was measured up to 11 d after application of chlorpyrifos. Plasma AChE reactivation in the presence of pyridine‐2–aldoxime methochloride (2–PAM) appeared to be a more sensitive indicator of exposure to chlorpyrifos than plasma ChE or BChE activity levels.