We have shown previously that axonal microtubules (MTs) are composite, consisting of two distinct domains that differ in their content of tyrosinated alpha-tubulin (tyr-tub). One domain is poor in tyr-tub and is situated at the minus end of the MT. The other domain is rich in tyr-tub and extends from the plus end of the tyr-tub-poor domain to the end of the MT. We have proposed that the spatial variation in the relative content of tyr-tub along individual MTs reflects corresponding variations in their dynamic properties. The present experiments have tested this hypothesis directly by microinjecting biotin-labeled tubulin (Bt-tub) into cultured sympathetic neurons and then quantifying the appearance of Bt-tub in the tyr-tub-rich and tyr-tub-poor polymer of the axon. Bt-tub appeared in axonal MTs with a half-life (t1/2) of approximately 2.2 hr. This time course reflected an average of two distinct components corresponding to the tyr-tub-rich and tyr-tub-poor polymer that had apparent t1/2 values of approximately 1.3 and 3.3 hr, respectively. In individual MTs, Bt-tub first appeared in the tyr-tub-rich domain and then only later appeared in the tyr-tub-poor domain. Also, the appearance of Bt-tub in the tyr-tub-rich polymer typically began precisely at its origin from the tyr-tub-poor domain, indicating that the tyr-tub-poor polymer is assembly-competent and nucleates the assembly of tyr-tub-rich polymer locally within the axon. The stability properties of axonal MTs are discussed in terms of organizing MT assembly locally in the axon and generating long MTs for growing long axons.