Of the many biologically isolated adeno-associated virus (AAV) serotypes, AAV1 and AAV6 share the highest degree of sequence homology, with only six different capsid residues. We compared the transduction efficiencies of rAAV1 and rAAV6 in primary polarized human airway epithelia and found significant differences in their abilities to transduce epithelia from the apical and basolateral membranes. rAAV1 transduction was ~10-fold higher than rAAV6 following apical infection, whereas rAAV6 transduction was ~10-fold higher than rAAV1 following basolateral infection. Furthermore, rAAV6 demonstrated significant polarity of transduction (100-fold; basolateral » apical), whereas rAAV1 transduced from both membranes with equal efficiency. To evaluate capsid residues responsible for the observed serotype differences, we mutated the six divergent amino acids either alone or in combination. Results from these studies demonstrated that capsid residues 418 and 531 most significantly controlled membrane polarity differences in transduction between serotypes, with the rAAV6-D418E/K531E mutant demonstrating decreased (~10-fold) basolateral transduction and the rAAV1-E418D/E531K mutant demonstrating a transduction polarity identical to rAAV6-WT (wild type). However, none of the rAAV6 mutants obtained apical transduction efficiencies of rAAV1-WT, suggesting that all six divergent capsid residues in AAV1 act in concert to improve apical transduction of HAE.