We have used the monoclonal antibody YL 1/2 (Tyr) specific for tyrosinated tubulin, and a polyclonal antibody (Glu) specific for detyrosinated tubulin to visualize the distribution of microtubules and microtubule assembly sites during axonal outgrowth. Cerebellar macroneurons growing in culture initially extend several short and thin neurites which have the potential to differentiate either as axons or dendrites (Ferreira and Caceres: Developmental Brain Research 49:205-213, 1989). At the onset of axonal outgrowth the Tyr antibody labels the minor neurites, the axon, and its growth cone, while the Glu antibody only shows immunoreactivity in the axonal shaft. After nocodazole treatment, the Tyr staining disappears, whereas that produced by the Glu antibody remains practically unchanged. When nocodazole was removed, tyrosinated microtubules reappeared first at the tip of the axon, in a more distal region than that occupied by detyrosinated microtubules; another focal site of tyrosinated tubulin incorporation was detected in the cell body. Incorporation of tyrosinated tubulin into growing axons was also studied after taxol treatment. After long incubation periods in the presence of taxol, the Tyr staining disappeared from the axon but remained in the cell body; however, immunoreactivity in this site was negative when the cells were preincubated in the presence of protein synthesis inhibitors. Release from taxol results in the reappearance of Tyr immunoreactivity at the distal end of the axon. Taken collectively, the present results indicate 1) that in cerebellar macroneurons axonal differentiation is accompanied by a temporal and spatial differentiation of microtubules and 2) that there is an active site of tyrosinated tubulin assembly at the tip of axonal processes, and they suggest that the highly tyrosinated domain in this region is a consequence of rapid microtubule turnover and tubulin tyrosine ligase activity.