The post-cytokinetic guard cells of Asplenium nidus display a prominent perinuclear microtubule system and a few microtubules under the periclinal walls. Afterwards, microtubules appear on the whole surface of the ventral wall, whereas those below the periclinal walls proliferate and tend to become parallel with the ventral wall. The perinuclear microtubules gradually diminish but persist in later stages of guard cell differentiation. In post-cytokinetic guard cells, actin is found in the perinuclear cytoplasm and in the cortical cytoplasm lining all the walls. In differentiating guard cells, the following cortical microtubules and actin filament ‘systems’ appear in succession: (a) radial microtubule and actin filament arrays beneath the periclinal walls converging on the stomatal pore region, (b) anticlinal microtubule bundles, which are co-localized with actin filaments, along the ventral wall outlining the region of the stomatal pore, (c) periclinal microtubules and actin filaments on the polar ventral wall ends. These cytoskeletal systems, except for the radial actin filaments, persist in advanced stages of guard cell differentiation. Instead of the radial actin filaments, a prominent actin filament reticulum is organized under the margins of the developing wall thickenings of the stomatal pore. In addition, an extensive endoplasmic actin filament reticulum develops around the plastids. It seems likely that the successive microtubule systems in guard cells are formed by putative microtubule organizing centres operating in a definite spatial and temporal succession. Guard cell morphogenesis is the outcome of a definite process, in which the cortical microtubule cytoskeleton plays the primary role, implicated in the deposition of cellulose microfibrils and probably of the local wall thickenings. Callose or a callose-like glucan is deposited on the whole surface of the nascent ventral wall and in the wall regions where thickenings are deposited. Finally, the guard cells of Asplenium assume a kidney shape and display polar hypostomatic swellings. Particular structural features established in guard cell mother cells affect guard cell morphogenesis.