This freeze-fracture study examines the development of myelinated fibers in the rat optic nerve. Axolemma of optic nerve fibers were studied before, during, and after myelination. At birth, the optic nerve is composed entirely of non-myelinated (premyelinated) axons, while in the adult, virtually all fibers acquire compact myelin. Myelination begins at 6-8 days postparturition and proceeds rapidly, such that by 28 days of age approximately 85% of the axons are myelinated. The axolemma of premyelinated fibers from 2-day-old animals exhibits an asymmetrical partitioning of intramembranous particles (IMPs) between E- and P-fracture faces; the E-face had approximately 125 particles/micron2 and the P-face approximately 550 particles/micron2. Particle densities for premyelinated axolemma from 8, 12, 14, 16, and 28-day-old nerves were similar to those observed at 2 days. Beginning at 8-12 days postnatal, definitive association between oligodendroglial processes and axons (termed 'ensheathed' fibers) was observed. At the time of glial ensheathment, there was a 50-100% increase in the number of P-face particles; in contrast, the E-face did not display an overall increase in particle density. In certain regions, however, localized aggregations of E-face particles were observed. IMPs on P-faces of ensheathed axons had a greater mean particle size and higher percentage of 'large' (greater than 9.6 nm) particles than did IMPs on the corresponding fracture face of premyelinated fibers. Myelinated axons from 14-16 day optic nerves displayed several differences from adult myelinated fibers. The P-face of the internodal axolemma had approximately 45% fewer particles than that of adult internodal membrane, and the percentage of large IMPs on the P-face of the younger internodal membrane was approximately 50% of the value for adult internodal axolemma. E-faces of internodal axolemma from 14-16-day-old and adult animals had equivalent IMP densities and size distributions. The nodal region of myelinated axons from 14-16-day-old rats had fewer large particles on both E- and P-faces than did adult fibers, though particle densities on both fracture faces were similar for the two age groups. These studies demonstrate a clear reorganization of axon membrane structure concomitant with axo-glial ensheathment, followed by continued gradual axolemmal changes as myelination progresses.