Second-harmonic generation (SHG) microscopy can image noncentrosymmetric protein structures with high hyperpolarizability, such as collagen and microtubules ( 17– 19), but few other known protein structures. Recently, fluorescently labeled plus-end tracking proteins have been used to estimate microtubule polarity in fruit flies ( 16) but not yet in mammals. However, the requirement of thin, fixed sections for electron microscopy ( 10, 11) has limited successful application of the hook method to only a few tissue preparations ( 14, 15). This difference between axons and dendrites of cultured hippocampal neurons had been implicated as a milestone in the establishment of neuronal polarity ( 13). The hook method, applied on hippocampal cultures ( 10– 12), indicated that microtubule arrays have uniform polarity (≈100% plus-ends distal from soma) in neurites of nascent neurons, uniform polarity in mature axons, and mixed polarity (≈50% plus-ends and ≈50% minus-ends distal from soma) in mature proximal dendrites. Past studies of microtubule polarity used electron microscopy to visualize neurites that were lysed in a tubulin-containing buffer, which would decorate clockwise or counterclockwise “hooks” on microtubules depending on their polarity ( 9). Recently, great progress has been made in elucidating the numerous motor proteins, including the molecular and functional classification of their superfamilies ( 1, 7, 8), but few studies have characterized the microtubule organization in neurons, particularly in dendritic processes. To understand this directional trafficking ( 3– 6), it is essential to know both the motor proteins and the microtubule polarities involved. These cargoes are transported by molecular motors, such as kinesin and dynein, which tread unidirectionally on microtubules. Anterograde transport through neurites delivers cargoes that are produced in the soma, including postsynaptic densities, neurotransmitter receptors, ion channels, and specific mRNAs to dendritic locations, as well as components of presynaptic terminals, adhesion molecules, and mitochondria to axonal locations ( 1, 2). Active intracellular transport along microtubules is essential for establishing and maintaining the functions of axons and dendrites.