Application of organophosphorus (OP) insecticides to agricultural fields during the spring often overlaps the period of spawning, egg incubation, and larval stages of fish species such as walleye (Stizostedion vitreum). Because life stage can affect uptake, distribution, and effects of contaminants, our objective was to identify the most sensitive life stage of walleye to chlorpyrifos, a broad-spectrum OP insecticide. Prolarvae (yolk sac, endogenous feeding stage) were least sensitive to chlorpyrifos (median lethal concentration [LC50] = 225–316 μg/L), and postlarvae I (oil globule, exogenous feeding stage) were less sensitive (LC50 = 24–29 μg/L) than postlarvae II (oil globule absent; LC50 = 12–13 μg/L). The differences in sensitivity of the larval stages coincide with stages of gill development. Gill filaments were absent until the end of the prolarval stage, and development of secondary lamellae did not occur until the end of the postlarval I stage. Juvenile fish were less sensitive than postlarval stages, but did not differ significantly among the juvenile ages tested. The LC50s ranged from 37 to 45 μg/L for 30- and 90-d-old juvenile walleye, respectively. Larval walleye survived when cholinesterase (ChE) activity was inhibited by as much as 90%; however, 60- and 90-d-old juvenile walleye did not survive when ChE activity was inhibited more than 71%.