Many of the structural and functional differences between axons are thought to reflect underlying differences in the biochemical composition and dynamic aspects of the axonal cytoskeleton and cytomatrix. In this study we investigated how the composition of the 2 slow components of axonal transport, SCa and SCb, which convey the cytoskeleton and cytomatrix, differs in axons that are structurally and functionally distinct. For this comparison we analyzed axons of retinal ganglion cells in the optic nerve (ON), axons of dorsal root ganglion (DRG) cells, and axons of ventral motor neurons (VMN) in adult rats. 35S-Methionine-labeled proteins transported with the peak of SCa and SCb were analyzed using high-resolution 2-dimensional polyacrylamide gels (2D-PAGE) and fluorography, and the amounts of major SCa and SCb proteins were quantified. The polypeptide composition of both SCa and SCb was found to be largely similar in DRG and VMN axons, but major qualitative as well as quantitative differences between these axons and ON axons were found. Notable among these were higher ratios of neurofilament protein to tubulin in SCa in DRG and VMN axons compared to ON axons, and significantly larger amounts of 2 microtubule-associated proteins relative to tubulin in SCa of ON axons than in both VMN and DRG axons. Tubulin was the major SCb protein in VMN and DRG axons, but it was not present in SCb in ON axons. Additionally, relatively larger amounts of 2 metabolic enzymes, creatine phosphokinase and nerve-specific enolase, were present in SCb in ON axons than in DRG or VMN axons. The results indicate that significant biochemical heterogeneity among different types of axons can be identified by examining the slow components of axonal transport.