Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.