Microtubule-associated protein 1b (MAP1b) is expressed at especially high levels in neurons actively extending axons, and although it appears to be required for axon growth, the nature of its role is unknown. We reasoned that a detailed description of the localization of MAP1b in growing axons would help define how MAP1b participates in axon growth. Therefore, we have stained cultured sympathetic neurons with various antibodies against MAP1b, and then used digital image processing and analysis procedures to quantify MAP1b distribution, phosphorylation and association with microtubules (MTs) in actively elongating axons. MAP1b is present on MTs all along the axon. Quantitative analyses showed that MAP1b has a nonuniform distribution along growing axons. It is present at relatively low and constant levels along the axon shaft until approximately 130 microns from the axon tip, where the amount of MAP1b begins to increase sharply and reaches a peak close to the growth cone. The peak amount of MAP1b in the distal axon is an order of magnitude greater than the average amount in the axon shaft. The enrichment of MAP1b in the distal axon was observed for total MAP1b and assembled MAP1b, and was even more pronounced for phosphorylated MAP1b. This distribution pattern remains after correcting the relative amount of MAP1b along the axon for variations in axonal volume. Thus, the concentration of MAP1b in the distal axon exceeds by severalfold that in the rest of the axon. The amount of assembled MAP1b relative to the amount of MT polymer also varies along the axon, and is greatest distally near the growth cone. This pattern of MAP1b localization in axons focuses attention on the distal axon and growth cone as the principal sites of MAP1b function in axon growth. We discuss the possibility that MAP1b regulates MT dynamics in the distal axon so that it is properly coordinated with growth cone events involved in axon extension.