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In airway epithelia, the kinetics of recombinant adeno‐associated virus (AAV) transgene expression is slow, with negative practical implications for research and for translation into therapy. It has also been shown that the DNA minor groove‐binding agent Hoechst‐33342 can enhance AAV transgene expression. Thus, in the present work, Dickey et al. investigated the mechanisms underlying the Hoechst‐33342‐related augmentation of AAV‐mediated transgene expression in HT1080, COS‐7, mouse and human airway epithelial cells. Hoechst‐33342 significantly accelerated AAV transgene expression for all AAV serotypes studied, but only when given during or after transduction, suggesting an effect on transgene expression regulation. This effect was not seen when the promoter was RSV. Hoechst dramatically augments and accelerates AAV‐ mediated transgene expression in airway epithelia, and Hoechst activation of the CMV promoter is seen in plasmids, but is drastically enhanced in the context of AAV.