Binding of the sterol-specific probe filipin to developing optic nerve axonal membrane is spatially heterogeneous prior to association of glial cells with the axons. Experiments were performed using different sterol binding probes (filipin, tomatin, and saponin), at different temperatures (4 degrees C, 23 degrees C, and 37 degrees C), after incubation in different ionic conditions (10 mM Ca2+, 10 mM EGTA, and 20 mM Mg2+), to examine factors that may be responsible for this membrane heterogeneity in rat optic nerve. The patchy pattern of filipin binding is apparent with each sterol-specific probe, even prior to glial ensheathment, and is retained when membrane fluidity is increased at higher temperatures. Increased Ca2+ concentration increased membrane stability, and increased Mg2+ reduced the patchiness of filipin binding. After tannic acid staining, regions of the cytoskeleton are seen associated with the membrane via filaments extending from microtubules to the membrane, preferentially in regions where filipin interaction with the membrane is inhibited. The non-uniform interaction of filipin with the axolemma suggests an underlying heterogeneity in the sterol composition and stability of the membrane. Heterogeneity of premyelinated axonal membrane may provide an important formative influence in the differentiation of axons to their mature morphology and function.