Porcine tracheae maintained in culture were used in order to study the colonization by Mycoplasma hyopneumoniae. Rings excised from tracheae of newborn piglets were infected with M hyopneumoniae strain BQ 14 and, after different incubation times, were examined by light and electron microscopy. Non-infected tracheal mucosae maintained a normal appearance for several days. Infected tracheal rings showed progressive colonization with concomitant progressive damage to the mucosal surface. Early on during the infection, few mycoplasmas occurred over a ciliated epithelium. As the infection progressed, there was gradual loss of cilia; mycoplasmas tended to form microcolonies and to accumulate over the remaining ciliated cells. Mycoplasmas, first seen at the apex of the cilia, were then seen deeper in the inter-ciliary space; some were even seen in contact with microvilli. In histological investigation, the final stage of the infection was characterized by a marked destruction of the epithelium with exfoliation of the epithelial cells. Infected mucosae showed typical damage caused by M hyopneumoniae, namely reduction of ciliary activity after 5 days, loss of cilia, and sloughing of ciliated cells. Our data indicate that porcine tracheal organ culture can be advantageously used to study colonization by M hyopneumoniae.