During the course of four to 14 days of topical application of isoflurophate ointment to rabbit eyes, there was a gradual increase in the sensitivity to acetylcholine of the superior recti muscles of treated eyes in vitro in the absence but not in the presence of eserine. The development of increased sensitivity to acetylcholine was accelerated when isoflurophate was applied twice daily. This increased sensitivity to acetylcholine was maintained when, after two weeks of twice-daily isoflurophate treatment, the frequency of administration was decreased to two applications per week. Partial reversal of this effect occurred seven to 14 days after the last isoflurophate treatment.