Adventitious root and shoot formation was obtained from cotyledon fragments of chestnut (Castanea sativa Mill.) and these processes followed two phases. In a first stage after detachment of the embryonic axis, the cotyledon fragments in culture formed a cotyledon petiole, which elongated for about 6 d. Thereafter, root primordia arose at the tip of the cotyledon petioles, followed by normal root development. In some cases, the cotyledon petioles showed adventitious shoot regeneration from a nodular structure previously formed at the end of the petioles. The presence or absence of growth regulators did not significantly influence root regeneration, whereas cytokinins stimulated shoot formation. The processes of root and shoot differentiation were studied also at the histological level. Observation with a light microscope showed that the developing root apical meristems were connected with a vascular bundle of the cotyledon petiole. Similarly, shoot bud meristem connections were observed with vascular tissue inside the nodular structure.