Dynamic organization of the axonal cytoskeleton was investigated by analyzing slow axonal transport of tubulin and other major cytoskeletal proteins in the motor axons of rat sciatic nerve 1-4 weeks after injection of L-35S-methionine into the anterior horn area of L3-L5 lumbar spinal cord. A large proportion (50-65%) of tubulin transported in the axon was found to be insoluble when extracted with 1% Triton at 4 degrees C. This cold-insoluble tubulin was also resistant to other microtubule-destabilizing agents such as Ca2+, colchicine, and nocodazole, suggesting that it corresponded to the stably polymerized tubulin specific to the axon. From the cold-soluble fraction, microtubules containing a distinct set of associated proteins were recovered by the taxol-dependent procedure. Transport pattern of cold-soluble and -insoluble tubulin in this system showed a time-dependent broadening of the tubulin wave resulting in the appearance of a new faster wave enriched in cold-soluble tubulin. The slower and the faster waves of tubulin were defined as group V or slow component a (SCa) and group IV or slow component b (SCb), respectively, with respect to the 2 subcomponents of slow transport originally described in the optic system. However, compositions of groups IV and V in sciatic motor axons differed significantly from those of the optic system. Actin also exhibited a clear dual wave pattern of transport that coincided well with that of tubulin, indicating that both actin and tubulin were the major components of both groups IV and V.(ABSTRACT TRUNCATED AT 250 WORDS)