Fluorescence digital imaging microscopy was used to investigate the process of myelin formation by Schwann cells in neuronal cocultures. The uptake of the fluorescent ceramide analogue N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]D-erythro-sphingosine (C5-DMB-ceramide) and its return to the plasma membrane as the corresponding fluorescent sphingomyelin and galactocerebroside analogues were measured. Through observation of this process it was possible to determine the rate of lipid synthesis in myelin internodes. The highest rate of synthesis of fluorescent sphingomyelin and galactocerebroside analogues was observed between days 3 and 7 after induction of myelination. This rate was approximately 5-fold greater than the steady-state rate of synthesis in fully myelinated internodes and 10-fold higher than the rate observed prior to myelination. The internode diameter increased during the first 3 days of myelination, but this was followed by a reduction in diameter and then an increase until the myelin sheath formation was completed. Internodes were found to be heterogeneous in terms of lipid distribution, with fluorescence intensities ranging 5-fold in myelinating cultures. Additionally, the rate of lipid transport along the internode was slow since there was a quicker increase in fluorescence intensity near the cell body of the Schwann cell than near the nodes of Ranvier. The results show that fluorescence digital imaging microscopy can be used to study the process of myelin formation and to determine the rate of formation, lipid transport, and heterogeneity of the myelin membrane.