We studied the dynamics of the cortical microtubule (CMT) cytoskeleton during differentiation of axial secondary phloem elements in taproots and epicotyls of Aesculus hippocastanum L. (horse-chestnut) saplings. Indirect immunofluorescence microscopy of alpha-tubulin and transmission electron microscopy revealed that fusiform cambial cells possessed a reticulum of CMTs in which individual microtubules were randomly arranged. During differentiation of these cambial cell derivatives into secondary phloem cells, the CMTs were rearranged to become helically oriented, regardless of phloem cell type. Although helical CMTs were a persistent feature of all axial elements of the secondary phloem (sieve elements, companion cells, phloem parenchyma, and fiber-sclereids), some modifications of this arrangement occurred as cells differentiated. Thus, at late stages of cell differentiation, sieve elements possessed nearly transverse CMTs, pronounced bundling of CMTs was seen in phloem parenchyma, and the density of CMTs in the helical arrays of fibers increased markedly. Additionally, phloem parenchyma possessed rings of CMTs in association with developing pit areas. Aspects of the development and chemistry of cell walls were also examined during phloem cytodifferentiation.