It has previously been estimated that about 60% of the neurons in the chick's isthmo-optic nucleus die during development, since its total number of neurons decreases by this percentage. Theoretically, however, the decrease need not have been due to cell death, but could have been caused by either of two alternative possibilities: neurons might have migrated out of the nucleus, or they might have shrunk and therefore been misidentified as glial cells at later developmental stages. These possibilities have been tested, using horseradish peroxidase and tritiated thymidine as tracers, and both have been disproved. Hence, the 60% neuronal death is genuine.