A non-optimal receptor-binding specificity of avian influenza viruses is believed to hamper their replication in humans; however, the magnitude of this restriction remains undefined. Here we generated recombinant viruses, R1 and R2, that differed solely by two amino acids in the receptor-binding site of their hemagglutinin (HA). R1 harbored the original HA of the pandemic human virus A/Hong Kong/1/68 (H3N2), whereas R2 was the L226Q/S228G HA mutant with avian-virus-like receptor specificity. In differentiated cultures of human tracheo-bronchial epithelial cells, R1 preferentially infected non-ciliated cells, whereas R2 predominantly infected ciliated cells indicating that cell tropism was determined by the viral receptor specificity. In the course of multi-cycle replication in these cultures, R2 spread less efficiently and grew to 2-10-fold lower titers than did R1. These results for the first time estimate the level of receptor-dependent restriction of avian influenza viruses in human airway epithelium. They support a theory that alteration of the receptor specificity of an avian virus could facilitate its human-to-human transmission.