The interaction between Mycoplasma pneumonaie and the airway epithelium in vivo is complex and multifaceted. While multiple in vitro studies have been conducted studying this interaction with cell lines and animal cell and organ culture models, the interactions between M. pneumoniae and fully differentiated human airway epithelium in air-liquid interface culture remains unexplored. In the present study we investigated M. pneumoniae interactions with airway epithelium utilizing an air-liquid interface culture of differentiated normal human bronchial epithelial (NHBE) cells. Utilizing confocal microscopy we found that M. pneumoniae cells bound initially to ciliated epithelial cells, but colonization became more evenly distributed over the entire surface with time. M. pneumoniae infection resulted in stimulation of intercellular adhesion molecule 1 (ICAM-1) gene expression and soluble ICAM-1 production in this culture system.