Fluorescein diacetate (FDA) and propidium iodide (PI) were used as indicators of membrane integrity after Eimeria tenella sporozoites were treated with polyether ionophores. Flow cytometry was used to quantitate the structural and functional effects based on red or green fluorescence and shape index of the sporozoites. Two field isolates (FS119 and FS139) were essentially resistant to polyether ionophores administered under practical conditions, whereas a laboratory strain was considered sensitive. The shape of sporozoites changed after treatment with ionophores, and this could be detected by flow cytometry. Green-fluorescing cells declined in number as the membranes were compromised by ionophore treatment. Red-fluorescing cells increased as the compromised membranes allowed entry of PI to bind with the nucleic acids. These effects were generally slower to develop in ionophore-tolerant field isolates compared with the sensitive laboratory strain. The effect of lasalocid on FDA and PI uptake, change in shape of the sporozoites, and lysis of sporozoites was more rapid than that of monensin or salinomycin. The 2 field isolates responded at different rates to salinomycin and monensin. Flow cytometry was a sensitive and accurate instrument for analysis of the effects of ionophores on sensitive and resistant lines of coccidia.