Hamster tracheal organ cultures were infected with M. pneumoniae and examined sequentially by transmission and scanning electron microscopy to correlate surface with intracellular alterations. Infection was established by culture and the demonstration of morphologically compatible organisms on the mucosal surface. Ciliated epithelial cells developed vacuolization of the apical and subnuclear cytoplasm and eventually fragmented along planes formed by coalescing vacuoles. Non-ciliated cells showed apical swelling and loss of microvilli during the course of infection. After degeneration and sloughing of both ciliated and non-ciliated cells, a flattened layer of intact basal cells covered the submucosa. It is likely that progressive vacuolization of epithelial cells leads to exfoliation of both cells and cell fragments in M. pneumoniae infection. Since organisms frequently are associated with these exfoliated cells, their potential presence in sputum and lavage specimens could prove to be of diagnostic importance.