Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons. Reliable information about axonal MVBs under physiological and pathological conditions is needed for a realistic assessment of their functional roles in neurons. We provide a quantitative ultrastructural analysis of MVBs in the normal postnatal rat hypoglossal nerve and under a variety of experimental conditions. MVBs were about 50 times less frequent in axons than in neuronal cell bodies or dendrites. Five distinct types of MVBs were distinguished in axons, based on MVB size, electron density, and size of internal vesicles. Although target manipulations did not significantly change MVBs in axons, dystrophic conditions such as delayed fixation substantially increased the number of axonal MVBs. Radiolabeled brain- and glial-cell derived neurotrophic factors (BDNF and GDNF) injected into the tongue did not accumulate during retrograde axonal transport in MVBs, as determined by quantitative ultrastructural autoradiography, and confirmed by analysis of quantum dot-labeled BDNF. We conclude that for axonal transport, neurotrophic factors utilize small vesicles or endosomes that can be inconspicuous at transmission electron microscopic resolution, rather than MVBs. Previous reports of axonal MVBs may be based, in part, on artificial generation of such organelles in axons due to dystrophic conditions. J. Comp. Neurol. 514:641–657, 2009. © 2009 Wiley-Liss, Inc.