We used an in vitro model of differentiated tracheobronchial epithelium to analyze the susceptibility of different cell types to infection with rhinoviruses (RVs). Primary cells from control subjects were cultured in an air-liquid interface to form differentiated epithelia. Suprabasal and basal fractions were separated after trypsin digestion, and cell suspensions were infected with serotypes RV16 and RV1A. These cell fractions were analyzed for expression of viral capsid protein VP2 (flow cytometry), viral replication (real-time PCR), cytokeratin-14, and intercellular adhesion molecule-1 (ICAM-1). Compared with suprabasal fraction, basal cells had increased percentages of cells staining positive for VP2 (RV1A: 37.8% versus 9.1%, P < 0.01; RV16: 12.0 versus 3.0%, P < 0.05). The average number of viral RNA copies per cell was also higher in basal cells (2.2- and 2.4-fold increase in RV1A- and RV16-infected cells, respectively) compared with suprabasal cells. Furthermore, ICAM-1 was expressed by 33.3% of basal cells, compared with 8.1% of suprabasal cells (P < 0.05). Finally, in culture models of epithelial injury (detached suprabasal cells or scratched surface), there was significantly greater replication of RV1A compared with intact cell layer. These findings demonstrate that basal cells are more susceptible to RV infection than suprabasal cells. For major group RV, this may be in part due to increased expression of ICAM-1; however, minor group RV also replicated more effectively in basal cells. These results suggest the possibility that epithelial cell differentiation is associated with the maturation of antiviral defense mechanisms.