Normal guinea pig optic nerve heads were examined using the quick-freeze, deep-etch, rotary-shadow technique. This method visualized the three-dimensional organization of the cytoskeleton. The axon cytoplasm was composed of 20-25 nm in diameter microtubules (MTs) and 10-12 nm in diameter neurofilaments (NFs) with numerous cross-linkers. These cross-linkers spanned between adjacent NFs, adjacent MTs, and between NFs and MTs. The membranous organelles such as mitochondria, axoplasmic reticulum, or tubulo-vesicular structures also were attached to the NFs and MTs with the cross-linkers. Abundant intermediate filaments of 10-12 nm diameter within glial cell components segregated axon bundles from laminar collagen fibers. The laminar fibers contained interstitial collagens that were 30-60 nm in diameter with characteristic striae and sidearms, and 5-10 nm wide mesh-like structures connecting the collagen fibril bundles to a surrounding basal lamina.