Eucalyptus nitens Maiden (shining gum) is a frost-tolerant species of Eucalyptus that can be used as an alternative species to Eucalyptus globulus in some regions of Portugal where winter temperatures are too low. Seedlings and 1-yr-old shoot tips and nodes were used for micropropagation of E. nitens. The best multiplication rate (2.25) was obtained when seedling shoots (<15 mm) were cultured on a medium containing the major nutrients (at half-strength) and minor elements of Murashige and Skoog (1962) medium, the organics of De Fossard medium (De Fossard et al., 1974) and a combination of benzyladenine (0.9 μM) and 1-naphthaleneacetic acid (0.05 μM). Seedling cuttings (4-,8-, and 10-wk-old) rooted well on media containing several concentrations of 3-indolebutyric acid (4.9, 9.8, and 14.8 μM) or 3-indoleacetic acid (5.7, 11.4, and 17.1 μM), giving frequencies of root induction above 80%. With this type of explant, root formation was also found on basal medium without growth regulators. Rooting of in vitro-propagated shoots obtained from seedlings (8-wk-old) after four subcultures (every 3 wk) was more difficult, with the best results obtained on a medium containing 14.7 μM 3-indolebutyric acid (60.0% root induction). No root formation was achieved when shoots from 1-yr-old explants were used. After a period of 4 mo., 96.3% of the plants transferred to the greenhouse survived acclimatization.