Resistance involving penetrability of cotyledons of subterranean clover Trifolium subterraneum (Leguminosae) by the redlegged earth mite, Halotydeus destructor Tucker, was examined in this study using several approaches. Values of penetrability, presented as mechanical strength measured with a penetrometer with a tubulated probe, of cotyledons tested 11-13 days after seeding, from nine varieties of two subspecies of subterranean clover (T. subterraneum subsp. yanninicum and subsp. brachycalicinum) were negatively correlated with feeding damage caused by H. destructor in both 3 h pairwise choice tests in a Petri dish with detached cotyledons, and in 2 week multiple choice tests in containers with seedlings. Simulation tests with different artificial membranes (Parafilm and Gladwrap) making up sachets, containing 5% glucose as a feeding stimulant, indicated that within 3 h H. destructor preferred membranes with lower strength. Field-collected young adults had significantly higher physical fitness to feed and/or gather on the artificial membrane sachet containing 5% glucose, than those from the laboratory culture, when the sachet was compared with cotyledons of either resistant (DGI007) or susceptible (Dalkeith) varieties in the choice tests. Electron microscopic observations of mite infested cotyledons of the resistant variety (DGI007) reveal that upper epidermal cell walls around penetration holes are bent inwards. Anticlinal cell walls of palisade cells appear buckled following attack by H. destructor. The results from these studies were taken as further evidence for the involvement of mechanical factors in antixenotic resistance of subterranean clover cotyledons to H. destructor.