A study of lesion development in stems of Eucalyptus nitens following artificial inoculations with canker fungi was carried out on 16-year-old plantation trees. In a first trial cambium bark wounds on smooth- and rough-barked trees were inoculated with the mycelium of nine species of canker fungi, including Endothia gyrosa. In a second trial spores or mycelium of E. gyrosa were applied directly onto undamaged or superficially wounded bark surfaces. Infection subsequent to artificial inoculation via wounding (whatever the wounding technique or type of inoculum) resulted in significantly larger external lesions (mean lesion area up to 35.6 cm2 20 months after inoculation) on smooth bark compared with those on rough bark (up to 19.0 cm2). Microscopic studies of infected rough and smooth bark suggest that, once smooth bark is compromised by wounding and artificial inoculation, the particular anatomical structure of smooth bark may offer less mechanical resistance to postpenetration hyphal spread in comparison with rough bark. It is suggested that at a pre-penetration stage under natural conditions spores of E. gyrosa more easily infect rough bark via cracks associated with this type of bark but not present in smooth bark.