Myelin sheaths of rapidly growing rats were sequentially labeled with the 3H and 14C isotopes of leucine as precursors of protein synthesis. The two injections were separated by time intervals ranging from 2 to 12 d. Wallerian degeneration was initiated by sciatic nerve neurotomy at 2 or 10 d after the second injection of radioactivity. After 5 d of degeneration, myelin was purified and the ratio of isotopes was determined in the delipidated protein. Regardless of the order in which the two isotopes were administered, the relative recovery of radioactivity resultant from the second injection was greatly reduced in degenerating nerves compared with sham-operated controls. Radioactivity incorporated from the first injection was also reduced, but to a lesser extent. Consequently, the isotope ratio corresponding to the first/second injection was greater in degenerating nerves than in controls, and the ratio increased in proportion to the time interval separating the two injections. The magnitude of the effect of degeneration was only slightly greater when degeneration was initiated 2 d after the second injection than when initiated 10 d after the last injection. Consequently, myelin disintegration rather than diminished incorporation of radioactivity accounts for the losses of radioactivity. Furthermore, the pattern of myelin degeneration preferentially involves the last myelin to be formed.