A precise correlation exists between the organization of microtubules and the orientation of newly deposited wall microfibrils in the developing cotton fiber. Reorganization of the microtubule array results in subsequent changes in wall microfibril orientation. Disrupting arrays of microtubules with various chemical agents does not result in the deposition of randomly organized wall microfibrils. Treatment of ovule culture derived fibers with colchicine results in fiber cells with only a few, widely dispersed microtubules. In these cells microfibril orientation and location is influenced by the microtubules that remain. Treatment with taxol induces bundling of microtubules and results in the formation of localized wall thickening. Even as disrupted arrays, microtubules influence the location and orientation of newly synthesized microfibrils. In the complete absence of microtubules (with prolonged exposure to colchicine), microfibril orientation is not random, but rather appears as swirls of parallel microfibrils. This observation indicates that the cellulose synthesizing machinery must have some capability of inducing order to the microfibril array. Microfibril order, at least over small regions of the cell, can be maintained independent of microtubule function. Microtubules may be involved in maintaining that order on a cell-wide level or in changing orientation of microfibril deposition during development.